Immune response inducer

ABSTRACT

An immunity-inducing agent comprising as an effective ingredient a specific polypeptide is disclosed. These polypeptides were isolated, by the SEREX method using a cDNA library derived from canine testis and serum from a cancer-bearing dog, as a polypeptide which binds to an antibody existing specifically in serum derived from a cancer-bearing living body. The polypeptides can induce immunity in a living body and cause regression of a tumor in a cancer-bearing living body. Therefore, these polypeptides are especially effective as a therapeutic and/or prophylactic agent for a cancer(s).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.12/739,723 filed on Apr. 23, 2010, which is the National Stage of PCTInternational Application No. PCT/JP2008/069267, filed on Oct. 23, 2008,which claims priority to Patent Application Nos. 2007-277578,2007-277611, 2007-277240 and 2007-279113, filed in Japan on Oct. 25,2007, Oct. 25, 2007, Oct. 25, 2007 and Oct. 26, 2007, all of which arehereby expressly incorporated by reference into the present application.

TECHNICAL FIELD

The present invention relates to a novel immunity-inducing agent usefulas a therapeutic and/or prophylactic agent for a cancer(s).

BACKGROUND ART

Cancers are the commonest cause for death among all of the causes fordeath, and the therapies therefor are mainly surgical treatment incombination with radiotherapy and chemotherapy. In spite of thedevelopments of new surgical methods and discovery of new anti-canceragents in recent years, treatment results of cancers are not improvedvery much at present except for some cancers. In recent years, by virtueof the development in molecular biology and cancer immunology, cancerantigens recognized by cytotoxic T cells reactive with cancers, as wellas the genes encoding the cancer antigens, were identified, andexpectations for antigen-specific immunotherapies have been raised (seeNon-patent Literature 1). In immunotherapy, to reduce side effects, itis necessary that the peptide or protein recognized as the antigen existhardly in normal cells and exist specifically in cancer cells. In 1991,Boon et al. of Ludwig Institute in Belgium isolated human melanomaantigen MAGE 1 recognized by CD8-positive T cells by a cDNA-expressioncloning method using an autologous cancer cell line and cancer-reactiveT cells (see Non-patent Literature 2). Thereafter, the SEREX(serological identifications of antigens by recombinant expressioncloning) method, wherein tumor antigens recognized by antibodiesproduced in the living body of a cancer patient in response to thecancer of the patient himself are identified by application of a geneexpression cloning method, was reported (Non-patent Literature 3; PatentLiterature 1), and various cancer antigens have been isolated (seeNon-patent Literatures 4 to 9). Using a part thereof as targets,clinical tests for cancer immunotherapy have started.

On the other hand, as in human, a number of tumors such as mammary glandtumor and squamous cell carcinoma are known in dogs and cats, and theyrank high also in the statistics of diseases in dogs and cats. However,at present, no therapeutic, prophylactic or diagnostic agents existwhich are effective for cancers in dogs and cats. Most of tumors in dogsand cats are realized by owners only after they advance to grow bigger,and in many cases, it is already too late to visit a hospital to receivesurgical excision of the tumor or administration of a human drug (ananticancer preparation or the like), so that those dogs and cats dieshortly after the treatment. Under such circumstances, if therapeuticagents, prophylactic agents and diagnostic agents for cancers effectivefor dogs and cats become available, their uses for canine cancers areexpected to be developed.

-   Patent Literature 1: U.S. Pat. No. 5,698,396 B-   Non-patent Literature 1: Tsuyoshi Akiyoshi, Cancer and Chemotherapy,    24, 551-519 (1997)-   Non-patent Literature 2: Bruggen P. et al., Science, 254:1643-1647    (1991)-   Non-patent Literature 3: Proc. Natl. Acad. Sci. USA, 92:11810-11813    (1995)-   Non-patent Literature 4: Int. J. Cancer, 72:965-971 (1997)-   Non-patent Literature 5: Cancer Res., 58:1034-1041 (1998)-   Non-patent Literature 6: Int. J. Cancer, 29:652-658 (1998)-   Non-patent Literature 7: Int. J. Oncol., 14:703-708 (1999)-   Non-patent Literature 8: Cancer Res., 56:4766-4772 (1996)-   Non-patent Literature 9: Hum. Mol. Genet 6:33-39, 1997-   Non-patent Literature 10: Naokazu Inoue, Ryo Yamaguchi and Masahito    Ikawa, Protein, Nucleic Acid and Enzyme, Vol. 50, No. 11, 1405-1412-   Non-patent Literature 11: J Cell Sci. 115:1825-35-   Non-patent Literature 12: Blood. 95:1788-96-   Non-patent Literature 13: Mol Endocrinol. 9:243-54 (1995)-   Non-patent Literature 14: J Cell Biol. 145: 83-98 (1999)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a novelimmunity-inducing agent which is useful as a therapeutic and/orprophylactic agent for a cancer(s)

Means for Solving the Problems

The present inventors intensively studied to obtain a cDNA encoding aprotein which binds to an antibody existing in serum derived from acancer-bearing living body by the SEREX method using a cDNA libraryderived from canine testis and serum of a cancer-bearing dog, which cDNAwas used to prepare a polypeptide having the amino acid sequence shownin SEQ ID NO:2, a canine calmegin protein having the amino acid sequenceshown in SEQ ID NO:16, a canine centrosomal protein (which may behereinafter abbreviated as CEP) having the amino acid sequence shown inSEQ ID NO:26, and the canine thyroid hormone receptor interactor 11(which may be hereinafter described as “TRIP11”) having the amino acidsequence shown in SEQ ID NO:39. Further, based on a registered caninegene having a high homology to the canine CEP of the above-described SEQID NO:26, a canine CEP having the amino acid sequence shown in SEQ IDNO:28 was prepared. Further, based on a human gene homologous to theobtained gene, a polypeptide having the amino acid sequence shown in SEQID NO:4, a human calmegin protein having the amino acid sequence shownin SEQ ID NO:18, a human CEP having the amino acid sequence shown in SEQID NO:30, and a human TRIP11 having the amino acid sequence shown in SEQID NO:41 were prepared. The inventors then discovered that thesepolypeptides can induce immunocytes in a living body and causeregression of an already occurred tumor when administered to the livingbody, thereby completing the present invention.

That is, the present invention provides an immunity-inducing agentcomprising as an effective ingredient any one of the polypeptides (a) to(c) below, the polypeptide having an immunity-inducing activity, or asan effective ingredient a recombinant vector which comprises apolynucleotide encoding the polypeptide and is capable of expressing thepolypeptide in vivo: (a) a polypeptide consisting of not less than 7consecutive amino acids of the amino acid sequence shown in SEQ ID NO:2,4, 16, 18, 26, 28, 30, 39 or 41 in SEQUENCE LISTING; (b) a polypeptidehaving a homology of not less than 80% to the polypeptide (a) andconsisting of not less than 7 amino acids; and (c) a polypeptidecomprising the polypeptide (a) or (b) as a partial sequence thereof. Thepresent invention also provides a method for inducing immunity, themethod comprising administering to an individual an effective amount ofany one of the above-described polypeptides (a) to (c), the polypeptidehaving an immunity-inducing activity, or an effective amount of arecombinant vector which comprises a polynucleotide encoding thepolypeptide and is capable of expressing the polypeptide in vivo. Thepresent invention further provides a method for treatingantigen-presenting cells, the method comprising bringing any one of theabove-described polypeptides (a) to (c), the polypeptide having animmunity-inducing activity, into contact with antigen-presenting cells.The present invention further provides use of any one of theabove-described polypeptides (a) to (c), the polypeptide having animmunity-inducing activity, or a recombinant vector which comprises apolynucleotide encoding the polypeptide and is capable of expressing thepolypeptide in vivo, for production of an immunity-inducing agent.

Effect of the Invention

By the present invention, a novel immunity-inducing agent useful as atherapeutic and/or prophylactic agent for a cancer(s) was provided. Asindicated in the Examples below, the polypeptide used in the presentinvention can induce immunocytes in a cancer-bearing dog and also cancause reduction or regression of an already occurred tumor whenadministered to a cancer-bearing dog. Therefore, the polypeptide isuseful for therapy and prophylaxis of a cancer(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression pattern of the gene identified in ExampleA-1 in normal tissues and tumor cell lines. Reference numeral 1: theexpression pattern of the identified gene; Reference numeral 2: theexpression pattern of the GAPDH gene.

FIG. 2 shows the detection by Coomassie staining of the canine-derivedprotein produced in E. coli and purified in Example A, which protein wasidentified in the present invention. Reference numeral 3: the band forthe canine-derived protein of the present invention.

FIG. 3 shows the expression pattern of the calmegin gene identified inthe present invention in normal tissues and tumor cell lines. Referencenumeral 1: the expression pattern of the calmegin gene; Referencenumeral 2: the expression pattern of the GAPDH gene.

FIG. 4 shows the detection by Coomassie staining of the canine calmeginprotein, which is an example of the polypeptide used in the presentinvention, produced in E. coli and purified in Example B. Referencenumeral 3: the band for the canine calmegin protein.

FIG. 5 shows the expression pattern of the gene encoding the CEP proteinin normal tissues and tumor cell lines. Reference numeral 1: theexpression pattern of the gene encoding the CEP protein; Referencenumeral 2: the expression pattern of the GAPDH gene.

FIG. 6 shows the detection by Coomassie staining of the canine CEP ofSEQ ID NO:26, which is an example of the polypeptide used in the presentinvention, produced in E. coli and purified in Example C. Referencenumeral 3: the band for the canine CEP protein.

FIG. 7 shows the expression pattern of the gene encoding the TRIP11protein in normal tissues and tumor cell lines. Reference numeral 1: theexpression pattern of the gene encoding the TRIP11 protein; Referencenumeral 2: the expression pattern of the GAPDH gene.

FIG. 8 shows the detection by Coomassie staining of the canine TRIP11protein, which is one of the polypeptides used in the present invention,produced in E. coli and purified in Example D. Reference numeral 3: theband for the canine TRIP11 protein.

BEST MODE FOR CARRYING OUT THE INVENTION

The polypeptides contained in the immunity-inducing agents of thepresent invention as effective ingredients are as follows. It should benoted that the term “polypeptide” in the present invention means amolecule formed by peptide bonding of a plurality of amino acids, andincludes not only polypeptide molecules having large numbers of aminoacids constituting them, but also low molecular weight molecules havingsmall numbers of amino acids (oligopeptides) and full-length proteins.Thus, in the present invention, proteins consisting of the full lengthof SEQ ID NO:2, 4, 16, 18, 26, 28, 30, 39 or 41 are also included in“polypeptide”.

(a) A polypeptide which consists of not less than 7 consecutive aminoacids of a polypeptide having the amino acid sequence shown in SEQ IDNO:2, 4, 16, 18, 26, 28, 30, 39 or 41 in SEQUENCE LISTING and has animmunity-inducing activity.

(b) A polypeptide which has a homology of not less than 80% to thepolypeptide (a), consists of not less than 7 amino acids, and has animmunity-inducing activity.

(c) A polypeptide which comprises the polypeptide (a) or (b) as apartial sequence thereof and has an immunity-inducing activity.

It should be noted that the term “having the amino acid sequence” in thepresent invention means that amino acid residues are aligned in thatorder. Accordingly, for example, “a polypeptide having the amino acidsequence shown in SEQ ID NO:2” means a polypeptide having a size of 306amino acid residues, whose amino acid sequence is Met Ala Ala Leu . . .(snip) . . . Ile Thr Ser Pro as shown in SEQ ID NO:2. Further, “apolypeptide having the amino acid sequence shown in SEQ ID NO:2” may beabbreviated as “a polypeptide of SEQ ID NO:2”. This also applies to theterm “having the base sequence”.

As used herein, the term “immunity-inducing activity” means an abilityto induce immunocytes which secrete cytokines such as interferon in aliving body. Whether or not a polypeptide has an immunity-inducingactivity can be confirmed using, for example, the known ELISPOT assay.More particularly, for example, as described in the Examples below,cells such as peripheral blood mononuclear cells are obtained from aliving body to which a polypeptide whose immunity-inducing activity isto be evaluated was administered, which cells are then cocultivated withthe polypeptide, followed by measuring the amount of a cytokine producedby the cells using a specific antibody, thereby measuring the number ofimmunocytes in the cells, which enables evaluation of theimmunity-inducing activity. Further, as described in the Examples below,a recombinant polypeptide prepared based on the amino acid sequence ofSEQ ID NO:2, 4, 16, 18, 26, 28, 30, 39 or 41 can cause regression of atumor by its immunity-inducing activity when administered to acancer-bearing living body. Therefore, the above-describedimmunity-inducing activity can be evaluated also as the ability toinhibit the growth of cancer cells expressing the polypeptide of SEQ IDNO:2, 4, 16, 18, 26, 28, 30, 39 or 41 or to cause reduction ordisappearance of a cancer tissue (tumor) (hereinafter referred to as“anti-tumor activity”). The anti-tumor activity of a polypeptide can beconfirmed by, for example, observation of whether or not the tumor isreduced when the polypeptide was administered to a cancer-bearing livingbody, as more particularly described in the Examples below. Further, theanti-tumor activity of a polypeptide can be evaluated also byobservation of whether or not T cells stimulated with the polypeptide(that is, T cells brought into contact with antigen-presenting cellswhich present the polypeptide) show a cytotoxic activity against tumorcells in vitro. The contact between T cells and antigen-presenting cellscan be carried out by cocultivation of the both in a liquid medium, asmentioned below. Measurement of the cytotoxic activity can be carriedout by, for example, a known method called ⁵¹Cr release assay describedin Int. J. Cancer, 58:p317, 1994. In cases where a polypeptide is usedfor therapy and/or prophylaxis of a cancer(s), the evaluation of theimmunity-inducing activity is preferably carried out using theanti-tumor activity as an index, although the index is not restricted.

The amino acid sequence shown in SEQ ID NO:2 in SEQUENCE LISTING is theamino acid sequence of the polypeptide with unknown function isolated asa polypeptide which binds to an antibody existing specifically in serumderived from a cancer-bearing dog, which isolation was carried out bythe SEREX method using a canine testis-derived cDNA library and serum ofa cancer-bearing dog (see Example A-1). It is registered in the NCBIdatabase under Accession No. XP_535343 (protein) and Accession No.XM_535343 (coding gene), but its function has not been reported.Further, the amino acid sequence shown in SEQ ID NO:4 is an amino acidsequence of a human homologous factor of the polypeptide of SEQ ID NO:2isolated as described above. This human homologous factor is also aprotein whose function is unknown, which is registered in the NCBIdatabase under Accession No. NP_689873 (protein) and Accession No.NM_152660 (coding gene). The homology between them is 93% in terms ofbase sequence and 99% in terms of amino acid sequence.

The respective amino acid sequences shown in SEQ ID NOs:16 and 18 arethose of the calmegin protein isolated as a polypeptide and a humanhomologous factor thereof, which polypeptide binds to an antibodyexisting specifically in serum derived from a cancer-bearing dog, whichisolation was carried out by the SEREX method using a caninetestis-derived cDNA library and serum of a cancer-bearing dog (seeExample B-1). Calmegin was identified as a protein which is expressedspecifically at the time of differentiation of a spermatid, and it has achaperone activity in vitro. Since it is expressed only in testis anddisappears in a mature sperm, calmegin is considered to have a functionto fold proteins involved in differentiation of spermatid (Non-patentLiterature 10, Naokazu Inoue, Ryo Yamaguchi and Masahito Ikawa, Protein,Nucleic Acid and Enzyme, Vol. 50, No. 11, 1405-1412). However, there hasbeen no report showing that the protein is expressed in a cancer anduseful for therapy or prophylaxis thereof. The homology between thecanine calmegin gene and the human calmegin gene is 90% in terms of basesequence and 89% in terms of amino acid sequence.

The respective amino acid sequences shown in SEQ ID NOs:26, 28 and 30are those of the CEP isolated as a polypeptide, a canine factor having ahigh homology to the polypeptide and a human homologous factor of thepolypeptide, which polypeptide binds to an antibody existingspecifically in serum derived from a cancer-bearing dog, which isolationwas carried out by the SEREX method using a canine testis-derived cDNAlibrary and serum of a cancer-bearing dog (see Example C-1). CEP is aprotein which is required by the centrosome to control microtubules andalso involved in maturation of the centrosome. It is known thatchromosomal translocation frequently occurs in some ofmyeloproliferative disorders, and since the CEP gene exists at the pointwhere the translocation occurs, CEP is considered to have a certainrelationship with the disorders. However, there has been no reportshowing that the protein is expressed in a cancer and useful for therapyor prophylaxis thereof (Non-patent Literature 11: J Cell Sci.115:1825-35; Non-patent Literature 12: Blood. 95:1788-96). The homologybetween the canine CEP gene encoding the CEP of SEQ ID NO:26 and thehuman CEP gene is 87% in terms of base sequence and 84% in terms ofamino acid sequence.

The respective amino acid sequences shown in SEQ ID NOs:39 and 41 arethose of the TRIP11 protein isolated as a polypeptide and a humanhomologous factor thereof, which polypeptide binds to an antibodyexisting specifically in serum derived from a cancer-bearing dog, whichisolation was carried out by the SEREX method using a caninetestis-derived cDNA library and serum of a cancer-bearing dog (seeExample D-1). TRIP11 (thyroid hormone receptor interactor 11) was firstidentified as a factor which interacts with the thyroid hormone receptorβ, and its binding to Golgi bodies and microtubules also became evident,so that TRIP11 is considered to play a role in maintaining the shapes ofthese organelles. However, there has been no report showing that theprotein is expressed in a cancer and useful for therapy or prophylaxisthereof (Non-patent Literature 13, Mol Endocrinol. 9:243-54 (1995);Non-patent Literature 14, J Cell Biol. 145: 83-98 (1999)). The homologybetween the canine TRIP11 gene and the human TRIP11 gene is 88% in termsof base sequence and 86% in terms of amino acid sequence.

The polypeptide (a) consists of not less than 7 consecutive, preferablynot less than 9 consecutive amino acids of a polypeptide having theamino acid sequence shown in SEQ ID NO:2, 4, 16, 18, 26, 28, 30, 39 or41, and has an immunity-inducing activity. The polypeptide especiallypreferably has the amino acid sequence shown in SEQ ID NO:2, 4, 16, 18,26, 28, 30, 39 or 41. As known in the art, a polypeptide consists of notless than about 7 amino acid residues can exert its antigenicity. Thus,a polypeptide consists of not less than 7 consecutive amino acidresidues of the amino acid sequence shown in SEQ ID NO:2, 4, 16, 18, 26,28, 30, 39 or 41 can have an immunity-inducing activity, so that it canbe used for preparation of the immunity-inducing agent of the presentinvention. However, in view of the fact that antibodies produced againstantigenic substances in a living body are polyclonal antibodies, it isthought that an antigenic substance composed of larger number of aminoacid residues can induce more types of antibodies which can recognizevarious sites on the antigenic substance, thereby attaining higherimmunity-inducing activity. Therefore, in order to increase theimmunity-inducing activity, in the case of SEQ ID NO:2 or 4, the numberof the amino acid residues may be preferably not less than 30, morepreferably not less than 100, still more preferably not less than 200,still more preferably not less than 250. In the case of SEQ ID NO:16 or18, the number of the amino acid residues may be preferably not lessthan 30, more preferably not less than 100, still more preferably notless than 200, still more preferably not less than 400, still morepreferably not less than 550. In the case of SEQ ID NO:26, 28 or 30, thenumber of the amino acid residues may be preferably not less than 30,more preferably not less than 100, still more preferably not less than300, still more preferably not less than 600, still more preferably notless than 1000, still more preferably not less than 1500, still morepreferably not less than 2000. In the case of SEQ ID NO:39 or 41, thenumber of the amino acid residues may be preferably not less than 30,more preferably not less than 100, still more preferably not less than300, still more preferably not less than 600, still more preferably notless than 1000, still more preferably not less than 1500.

As a principle of immune induction by administration of a cancerantigenic polypeptide, the following process is known: the polypeptideis incorporated into an antigen-presenting cell and then degraded intosmaller fragments by peptidases in the cell, followed by presentation ofthe fragments on the surface of the cell. The fragments are thenrecognized by a cytotoxic T cell or the like, which selectively killscells presenting the antigen.

The size of the polypeptide presented on the surface of theantigen-presenting cell is relatively small and about 7 to 30 aminoacids. Therefore, from the view point of presenting thereof on thesurface of the antigen-presenting cell, a polypeptide consisting ofabout 7 to 30, preferably about 9 to 30 consecutive amino acids of theamino acid sequence shown in SEQ ID NO:2, 4, 16, 18, 26, 28, 30, 39 or41 is sufficient as the above-described polypeptide (a). In some cases,these relatively small polypeptides are presented directly on thesurface of the antigen-presenting cells without incorporation thereofinto the antigen-presenting cells.

However, as described above, since a polypeptide incorporated into anantigen-presenting cell is cleaved at random sites by peptidases in thecell to yield various polypeptide fragments, which are then presented onthe surface of the antigen-presenting cell, administration of a largepolypeptide such as the entire region of SEQ ID NO:2, 4, 16, 18, 26, 28,30, 39 or 41 inevitably causes production of polypeptide fragments bydegradation thereof in the antigen-presenting cell, which fragments areeffective for immune induction via the antigen-presenting cell.Therefore, for immune induction via antigen-presenting cells, a largepolypeptide can also be preferably used. In the case of SEQ ID NO:2 or4, the number of the amino acids may be preferably not less than 30,more preferably not less than 100, still more preferably not less than200, still more preferably not less than 250. In the case of SEQ IDNO:16 or 18, the number of the amino acids may be preferably not lessthan 30, more preferably not less than 100, still more preferably notless than 200, still more preferably not less than 400, still morepreferably not less than 550. In the case of SEQ ID NO:26, 28 or 30, thenumber of the amino acids may be preferably not less than 30, morepreferably not less than 100, still more preferably not less than 300,still more preferably not less than 600, still more preferably not lessthan 1000, still more preferably not less than 1500, still morepreferably not less than 2000. In the case of SEQ ID NO:39 or 41, thenumber of the amino acids may be preferably not less than 30, morepreferably not less than 100, still more preferably not less than 300,still more preferably not less than 600, still more preferably not lessthan 1000, still more preferably not less than 1500.

The above-described polypeptide (b) is the same polypeptide as theabove-described polypeptide (a) except that a small number of amino acidresidues are substituted, deleted and/or inserted, which has a homologyof not less than 80%, preferably not less than 90%, more preferably notless than 95%, still more preferably not less than 98% to the originalsequence, and has an immunity-inducing activity. It is well known in theart that, in general, there are cases where a protein antigen retainssubstantially the same antigenicity as the original even if the aminoacid sequence of the protein is modified such that a small number ofamino acids are substituted, deleted and/or inserted. Therefore, sincethe above-described polypeptide (b) may also exert an immunity-inducingactivity, it can be used for preparation of the immunity-inducing agentof the present invention. Further, the above-described polypeptide (b)is also preferably the same polypeptide as one having the amino acidsequence shown in SEQ ID NO:2, 4, 16, 18, 26, 28, 30, 39 or 41 exceptthat one or several amino acid residues are substituted, deleted and/orinserted.

As used herein, the term “homology” of amino acid sequences means avalue expressed in percentage which is calculated by aligning two aminoacid sequences to be compared such that the number of matched amino acidresidues is the maximum, and dividing the number of the matched aminoacid residues by the number of the total amino acid residues. When theabove-described alignment is carried out, a gap(s) is/are inserted intoone or both of the two sequences to be compared as required. Suchalignment of sequences can be carried out using a well-known programsuch as BLAST, FASTA or CLUSTAL W. When a gap(s) is/are inserted, theabove-described number of the total amino acid residues is calculated bycounting one gap as one amino acid residue. When the thus countednumbers of the total amino acid residues are different between the twosequences to be compared, the homology (%) is calculated by dividing thenumber of matched amino acid residues by the number of the total aminoacid residues in the longer sequence.

The 20 types of amino acids constituting the naturally occurringproteins may be classified into groups each of which has similarproperties, for example, into neutral amino acids with side chainshaving low polarity (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral aminoacids having hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys),acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His) andaromatic amino acids (Phe, Tyr, Trp). It is known that, in most cases,substitutions of amino acids within the same group do not change theproperties of the polypeptide. Therefore, in cases where amino acidresidue(s) in the above described polypeptide (a) in the presentinvention is/are substituted, the probability that the immunity-inducingactivity can be maintained may be made high by conducting thesubstitution(s) within the same group.

The above-described polypeptide (c) comprises the above-describedpolypeptide (a) or (b) as a partial sequence and has animmunity-inducing activity. That is, the polypeptide (c) hasanother/other amino acid(s) or polypeptide(s) added at one or both endsof the polypeptide (a) or (b), and has an immunity-inducing activity.Such a polypeptide can also be used for preparation of theimmunity-inducing agent of the present invention.

For example, the above-described polypeptides can be synthesized by achemical synthesis method such as the Fmoc method(fluorenylmethylcarbonyl method) or the tBoc method (t-butyloxycarbonylmethod). Further, they can be synthesized by conventional methods usingvarious commercially available peptide synthesizers. Further, thepolypeptide of interest can be obtained by a known genetic engineeringmethod wherein a polynucleotide encoding the above-described polypeptideis prepared and incorporated into an expression vector, which is thenintroduced into a host cell, in which the polypeptide is produced.

The polynucleotide encoding the above-described polypeptide can beeasily prepared by a known genetic engineering method or a conventionalmethod using a commercially available nucleic acid synthesizer. Forexample, DNA having the base sequence of SEQ ID NO:1, 15, 25, 27 or 38can be prepared by carrying out PCR using the chromosomal DNA or a cDNAlibrary of a dog as a template and using a pair of primers designed suchthat the primers can amplify the base sequence described in SEQ ID NO:1,15, 25, 27 or 38, respectively. DNA having the base sequence of SEQ IDNO:3, 17, 29 or 40 can be prepared similarly by using as theabove-described template the human chromosomal DNA or a cDNA library.Conditions for the PCR reaction can be selected as appropriate, andexamples of the conditions include, but are not limited to, thosewherein a cycle comprising the reaction steps of 94° C. for 30 seconds(denaturing), 55° C. for 30 seconds to 1 minute (annealing), and 72° C.for 2 minutes (extension) is repeated, for example, 30 times, followedby allowing the reaction to proceed at 72° C. for 7 minutes. Further, adesired DNA can be isolated by preparing an appropriate probe or primerbased on the information of the base sequence and the amino acidsequence shown in SEQ ID NOs:1 to 4, 15 to 18, 25 to 30, 38 to 41 inSEQUENCE LISTING of the present specification and then using the probeor primer for screening of a cDNA library from a dog or a human. ThecDNA library is preferably prepared from cells, an organ or a tissueexpressing the protein of SEQ ID NO:2, 4, 16, 18, 26, 28, 30, 39 or 41.Operations such as the above-described preparation of a probe or aprimer, construction of a cDNA library, screening of a cDNA library andcloning of a gene of interest are known to those skilled in the art, andcan be carried out according to, for example, Molecular Cloning, 2nd Ed.or Current Protocols in Molecular Biology. From the thus obtained DNA,DNA encoding the above-described polypeptide (a) can be obtained.Further, since codons encoding each amino acid are known, the basesequence of a polynucleotide encoding a specific amino acid sequence canbe easily specified. Therefore, the base sequences of polynucleotidesencoding the above-described polypeptide (b) and polypeptide (c) canalso be easily specified, so that such polynucleotides can also beeasily synthesized using a commercially available nucleic acidsynthesizer according to a conventional method.

The above-described host cells are not restricted as long as they canexpress the above-described polypeptide, and examples thereof include,but are not limited to, prokaryotic cells such as E. coli; andeukaryotic cells such as mammalian cultured cells including monkeykidney cells COS 1 and Chinese hamster ovary cells CHO, budding yeast,fission yeast, silkworm cells, and Xenopus laevis egg cells.

In cases where prokaryotic cells are used as the host cells, anexpression vector having the origin that enables its replication in aprokaryotic cell, a promoter, a ribosome binding site, a DNA cloningsite, a terminator and the like is used as the expression vector.Examples of the expression vector for E. coli include the pUC system,pBluescript II, pET expression system and pGEX expression system. Byincorporating DNA encoding the above-described polypeptide into such anexpression vector and transforming prokaryotic host cells with thevector, followed by culturing the obtained transformant, the polypeptideencoded by the above-described DNA can be expressed in the prokaryotichost cells. In this case, the polypeptide can also be expressed as afusion protein with another protein.

In cases where eukaryotic cells are used as the host cells, anexpression vector for eukaryotic cells having a promoter, splicing site,poly(A) addition site and the like is used as the expression vector.Examples of such an expression vector include pKA1, pCDM8, pSVK3, pMSG,pSVL, pBK-CMV, pBK-RSV, the EBV vector, pRS, pcDNA3, pMSG and pYES2. Inthe same manner as described above, by incorporating DNA encoding theabove-described polypeptide into such an expression vector andtransforming eukaryotic host cells with the vector, followed byculturing the obtained transformant, the polypeptide encoded by theabove-described DNA can be expressed in the eukaryotic host cells. Incases where pIND/V5-His, pFLAG-CMV-2, pEGFP-N1 or pEGFP-C1 was used asthe expression vector, the above-described polypeptide can be expressedas a fusion protein having various added tags such as His tag, FLAG tag,myc tag, HA tag or GFP.

Introduction of the expression vector to the host cells can be carriedout using a well-known method such as electroporation, the calciumphosphate method, the liposome method or the DEAE dextran method.

Isolation and purification of a polypeptide of interest from the hostcells can be carried out by a combination of known separationoperations. Examples of the operations include, but are not limited to,treatment by a denaturant such as urea or by a surfactant;ultrasonication treatment; enzyme digestion; salting-out and solventfractional precipitation; dialysis; centrifugation; ultrafiltration; gelfiltration; SDS-PAGE; isoelectric focusing; ion-exchange chromatography;hydrophobic chromatography; affinity chromatography; and reversed-phasechromatography.

The polypeptides obtained by the above method include, as mentionedabove, those in the form of a fusion protein with another arbitraryprotein. Examples thereof include fusion proteins with glutathionS-transferase (GST) and with a His tag. Such a polypeptide in the formof a fusion protein is also included within the scope of the presentinvention as the above-described polypeptide (c). Further, in somecases, a polypeptide expressed in a transformed cell is modified invarious ways in the cell after translation thereof. Such a polypeptidehaving a post-translational modification is also included within thescope of the present invention as long as it has an immunity-inducingactivity. Examples of such a post-translational modification includeelimination of N-terminus methionine, N-terminus acetylation,glycosylation, limited degradation by an intracellular protease,myristoylation, isoprenylation and phosphorylation.

As described concretely in the following Examples, the above-describedpolypeptide having an immunity-inducing activity can cause regression ofan already occurred tumor when administered to a cancer-bearing livingbody. Therefore, the immunity-inducing agent of the present inventioncan be used as a therapeutic and/or prophylactic agent for a cancer(s).In this case, cancers to be treated are those expressing the geneencoding the polypeptide of SEQ ID NO:2 or 4, and examples thereofinclude, but are not limited to, brain tumor; squamous cell carcinomasof head, neck, lung, uterus and esophagus; melanoma; adenocarcinomas oflung, breast and uterus; renal cancer; malignant mixed tumor;hepatocellular carcinoma; basal cell carcinoma; acanthomatous epulis;intraoral tumor; perianal adenocarcinoma; anal sac tumor; anal sacapocrine carcinoma; Sertoli cell tumor; vulva cancer; sebaceousadenocarcinoma; sebaceous epithelioma; sebaceous adenoma; sweat glandcarcinoma; intranasal adenocarcinoma; nasal adenocarcinoma; thyroidcancer; colon cancer; bronchial adenocarcinoma; adenocarcinoma; ductalcarcinoma; mammary adenocarcinoma; combined mammary adenocarcinoma;mammary gland malignant mixed tumor; intraductal papillaryadenocarcinoma; fibrosarcoma; hemangiopericytoma; osteosarcoma;chondrosarcoma; soft tissue sarcoma; histiocytic sarcoma; myxosarcoma;undifferentiated sarcoma; lung cancer; mastocytoma; cutaneous leiomyoma;intra-abdominal leiomyoma; leiomyoma; chronic lymphocytic leukemia;lymphoma; gastrointestinal lymphoma; digestive organ lymphoma; smallcell or medium cell lymphoma; adrenomedullary tumor; granulosa celltumor; pheochromocytoma; bladder cancer (transitional cell carcinoma);suppurative inflammation; intra-abdominal liver tumor; liver cancer;plasmacytoma; malignant hemangiopericytoma; angiosarcoma; anal sacadenocarcinoma; oral cancer; metastatic malignant melanoma; amelanoticmalignant melanoma; cutaneous malignant melanoma; malignantmyoepithelioma; malignant seminoma; seminoma; adenocarcinoma of thelarge intestine; gastric adenocarcinoma; low-grade sebaceous carcinoma;ceruminous adenocarcinoma; apocrine carcinoma; poorly differentiatedapocrine sweat gland carcinoma; malignant fibrous histiocytoma; multiplemyeloma; mesenchymal malignant tumor; liposarcoma; osteosarcoma; sarcomaof unknown origin; soft part sarcoma (spindle cell tumor); poorlydifferentiated sarcoma; synovial sarcoma; angiosarcoma; metastaticmalignant epithelioma; tubular mammary adenocarcinoma; mammary ductalcarcinoma; inflammatory breast cancer; germinoma; leukemia; invasivetrichoepithelioma; medium cell lymphoma; multicentric lymphoma;osteosarcoma (mammary gland); mastocytoma (Patnaik II type); mastocytoma(Grade II); and leiomyosarcoma. The animals to be treated are mammals,especially preferably humans, dogs and cats.

The administration route of the immunity-inducing agent of the presentinvention to a living body may be either oral administration orparenteral administration, and is preferably parenteral administrationsuch as intramuscular administration, subcutaneous administration,intravenous administration or intraarterial administration. In caseswhere the immunity-inducing agent is used for therapy of a cancer, itmay be administered to a regional lymph node in the vicinity of thetumor to be treated, as described in the Examples below, in order toenhance its anticancer activity. The dose may be any dose as long as thedose is effective for immune induction, and in cases where the agent isused for therapy and/or prophylaxis of a cancer, the dose may be oneeffective for therapy and/or prophylaxis of the cancer. The doseeffective for therapy and/or prophylaxis of a cancer is appropriatelyselected depending on the size of the tumor, the symptom and the like,and usually, 0.0001 μg to 1000 μg, preferably 0.001 μg to 1000 μg of theagent in terms of the effective ingredient may be administered once orin several times per day per animal to be treated. The agent ispreferably administered in several times, every several days to severalmonths. As concretely shown in the Examples below, the immunity-inducingagent of the present invention can cause regression of an alreadyoccurred tumor. Therefore, since the agent can exert its anticanceractivity also against a small number of cancer cells in the early stage,development or recurrence of the cancer can be prevented by using theagent before development of a cancer or after therapy for a cancer. Thatis, the immunity-inducing agent of the present invention is effectivefor both therapy and prophylaxis of a cancer.

The immunity-inducing agent of the present invention may contain only apolypeptide or may be formulated by mixing as appropriate with anadditive such as a pharmaceutically acceptable carrier, diluent orvehicle suitable for each administration mode. Formulation methods andadditives which may be used are well-known in the field of formulationof pharmaceuticals, and any of the methods and additives may be used.Specific examples of the additive include, but are not limited to,diluents such as physiological buffer solutions; vehicles such assucrose, lactose, corn starch, calcium phosphate, sorbitol and glycine;binders such as syrup, gelatin, gum arabic, sorbitol, polyvinyl chlorideand tragacanth; and lubricants such as magnesium stearate, polyethyleneglycol, talc and silica. Examples of the formulation include oralpreparations such as tablets, capsules, granules, powders and syrups;and parenteral preparations such as inhalants, injection solutions,suppositories and solutions. These formulations may be prepared bycommonly known production methods.

The immunity-inducing agent of the present invention may be used incombination with an immunoenhancer capable of enhancing the immuneresponse in a living body. The immunoenhancer may be contained in theimmunity-inducing agent of the present invention or administered as aseparate composition to a patient in combination with theimmunity-inducing agent of the present invention.

Examples of the above-described immunoenhancer include adjuvants.Adjuvants can enhance the immune response by providing a reservoir ofantigen (extracellularly or within macrophages), activating macrophagesand stimulating specific sets of lymphocytes, thereby enhancing theanticancer activity. Therefore, especially in cases where theimmunity-inducing agent of the present invention is used for therapyand/or prophylaxis of a cancer, the immunity-inducing agent preferablycomprises an adjuvant, in addition to the above-described polypeptide asan effective ingredient. Many types of adjuvants are well-known in theart, and any of these adjuvants may be used. Specific examples of theadjuvants include MPL (SmithKline Beecham) and homologues of Salmonellaminnesota Re 595 lipopolysaccharide obtained after purification and acidhydrolysis of the lipopolysaccharide; QS21 (SmithKline Beecham), pureQA-21 saponin purified from extract of Quillja saponaria; DQS21described in WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS-18 andQS-L1 (So and 10 others, “Molecules and cells”, 1997, Vol. 7, p.178-186); Freund's incomplete adjuvant; Freund's complete adjuvant;vitamin E; Montanide; alum; CpG oligonucleotides (see, for example,Kreig and 7 others, “Nature”, Vol. 374, p. 546-549); poly-I:C andderivatives thereof (e.g., poly ICLC); and various water in oilemulsions prepared from biodegradable oils such as squalene and/ortocopherol. Among these, Freund's incomplete adjuvant; Montanide;poly-I:C and derivatives thereof, and CpG oligonucleotides arepreferred. The mixing ratio between the above-described adjuvant andpolypeptide is typically about 1:10 to 10:1, preferably about 1:5 to5:1, more preferably about 1:1. However, the adjuvant is not limited tothe above-described examples, and adjuvants known in the art other thanthe above-described ones (for example, see Goding, “MonoclonalAntibodies: Principles and Practice”, 2nd edition, 1986) may be usedwhen the immunity-inducing agent of the present invention isadministered. Preparation methods for mixtures or emulsions of apolypeptide and an adjuvant are well-known to those skilled in the artof vaccination.

Further, in addition to the above-described adjuvants, factors thatstimulate the immune response of the subject may be used as theabove-described immunoenhancer. For example, various cytokines having aproperty to stimulate lymphocytes and/or antigen-presenting cells may beused as the immunoenhancer in combination with the immunity-inducingagent of the present invention. A number of such cytokines capable ofenhancing the immune response are known to those skilled in the art, andexamples thereof include, but are not limited to, interleukin-12(IL-12), GM-CSF, IL-18, interferon-α, interferon-β, interferon-ω,interferon-γ, and Flt3 ligand, which have been shown to promote theprophylactic action of vaccines. Such factors may also be used as theabove-described immunoenhancer, and can be contained in theimmunity-inducing agent of the present invention, or can be prepared asa separate composition to be administered to a patient in combinationwith the immunity-inducing agent of the present invention.

Further, by bringing the above-described polypeptide into contact withantigen-presenting cells in vitro, the antigen-presenting cells can bemade to present the polypeptide. That is, the above-describedpolypeptides (a) to (c) can be used as agents for treatingantigen-presenting cells. As the antigen-presenting cells, dendriticcells or B cells, which have MHC class I molecules, may preferably beemployed. Various MHC class I molecules have been identified andwell-known. MHC molecules in human are called HLA. Examples of HLA classI molecules include HLA-A, HLA-B and HLA-C, more specifically, HLA-A1,HLA-A0201, HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA-A11, HLA-A24,HLA-A31, HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401 andHLA-Cw0602.

The dendritic cells or B cells having MHC class I molecules can beprepared from peripheral blood by a well-known method. For example,tumor-specific dendritic cells can be induced by inducing dendriticcells from bone marrow, umbilical cord blood or patient's peripheralblood using granulocyte-macrophage colony-stimulating factor (GM-CSF)and IL-3 (or IL-4), and then adding a tumor-related peptide to theculture system. By administering an effective amount of such dendriticcells, a response desired for therapy of a cancer can be induced. As thecells to be used, bone marrow or umbilical cord blood donated by ahealthy individual, or bone marrow, peripheral blood or the like fromthe patient himself may be used. When autologous cells of the patientare used, high safety can be attained and serious side effects areexpected to be avoided. The peripheral blood or bone marrow may be afresh sample, cold-stored sample or frozen sample. As for the peripheralblood, whole blood may be cultured or the leukocyte components alone maybe separated and cultured, and the latter is efficient and thuspreferred. Further, among the leukocyte components, mononuclear cellsmay be separated. In cases where the cells are originated from bonemarrow or umbilical cord blood, the whole cells constituting the bonemarrow may be cultured, or mononuclear cells may be separated therefromand cultured. Peripheral blood, the leukocyte components thereof andbone marrow cells contain mononuclear cells, hematopoietic stem cellsand immature dendritic cells, from which dendritic cells are originated,and also CD4-positive cells and the like. As for the cytokine to beused, the production method thereof is not restricted andnaturally-occurring or recombinant cytokine or the like may be employedas long as its safety and physiological activity have been confirmed.Preferably, a preparation with assured quality for medical use is usedin a minimum necessary amount. The concentration of the cytokine(s) tobe added is not restricted as long as the dendritic cells are induced,and usually, the total concentration of the cytokine(s) is preferablyabout 10 to 1000 ng/mL, more preferably about 20 to 500 ng/mL. Theculture may be carried out using a well-known medium usually used forthe culture of leukocytes. The culturing temperature is not restrictedas long as the proliferation of the leukocytes is attained, and about37° C. which is the body temperature of human is most preferred. Theatmospheric environment during the culturing is not restricted as longas the proliferation of the leukocytes is attained, and to flow 5% CO₂is preferred. The culturing period is not restricted as long as thenecessary number of the cells is induced, and is usually 3 days to 2weeks. As for the apparatuses used for separation and culturing of thecells, appropriate apparatuses, preferably those whose safety whenapplied to medical uses have been confirmed, and whose operations arestable and simple, may be employed. Particularly, as for thecell-culturing apparatus, not only the general vessels such as a Petridish, flask and bottle, but also a layer type vessel, multistage vessel,roller bottle, spinner type bottle, bag type culturing vessel, hollowfiber column and the like may be used.

Bringing the above-described peptide of the present invention intocontact with the antigen presenting cells in vitro may be carried out bya well-known method. For example, it may be carried out by culturing theantigen-presenting cells in a culture medium containing theabove-described polypeptide. The concentration of the peptide in themedium is not restricted, and usually about 1 μg/ml to 100 μg/ml,preferably about 5 μg/ml to 20 μg/ml. The cell density during theculturing is not restricted and is usually about 10³ cells/ml to 10⁷cells/ml, preferably about 5×10⁴ cells/ml to 5×10⁶ cells/ml. Theculturing may be carried out according to a conventional method, and ispreferably carried out at 37° C. under atmosphere of 5% CO₂. The maximumlength of the peptide which can be presented on the surface of theantigen-presenting cells is usually about 30 amino acid residues.Therefore, in cases where the antigen-presenting cells are brought intocontact with the polypeptide in vitro, the polypeptide may be preparedsuch that its length is not more than about 30 amino acid residues.

By culturing the antigen-presenting cells in the coexistence of theabove-described polypeptide, the polypeptide is incorporated into MHCmolecules of the antigen-presenting cells and presented on the surfaceof the antigen-presenting cells. Therefore, using the above-describedpolypeptide, isolated antigen-presenting cells containing the complexbetween the polypeptide and the MHC molecule can be prepared. Suchantigen-presenting cells can present the polypeptide against T cells invivo or in vitro, and induce, and allow proliferation of, cytotoxic Tcells specific to the polypeptide.

By bringing the antigen-presenting cells prepared as described abovehaving the complex between the above-described polypeptide and the MHCmolecule into contact with T cells in vitro, cytotoxic T cells specificto the polypeptide can be induced and allowed to proliferate. This maybe carried out by cocultivating the above-described antigen-presentingcells and T cells in a liquid medium. For example, it may be attained bysuspending the antigen-presenting cells in a liquid medium, placing thesuspension in vessels such as wells of a microplate, adding thereto Tcells and then culturing the cells. The mixing ratio of theantigen-presenting cells to the T cells in the cocultivation is notrestricted, and is usually about 1:1 to 1:100, preferably about 1:5 to1:20 in terms of the number of cells. The density of theantigen-presenting cells suspended in the liquid medium is notrestricted, and is usually about 100 to 10,000,000 cells/ml, preferablyabout 10,000 to 1,000,000 cells/ml. The cocultivation is preferablycarried out at 37° C. under atmosphere of 5% CO₂ in accordance with aconventional method. The culturing time is not restricted, and isusually 2 days to 3 weeks, preferably about 4 days to 2 weeks. Thecocultivation is preferably carried out in the presence of one or moreinterleukins such as IL-2, IL-6, IL-7 and IL-12. In this case, theconcentration of IL-2 and IL-7 is usually about 5 U/ml to 20 U/ml, theconcentration of IL-6 is usually about 500 U/ml to 2000 U/ml, and theconcentration of IL-12 is usually about 5 ng/ml to 20 ng/ml, but theconcentrations of the interleukins are not restricted thereto. Theabove-described cocultivation may be repeated once to several timesadding fresh antigen-presenting cells. For example, the operation ofdiscarding the culture supernatant after the cocultivation and adding afresh suspension of antigen-presenting cells to further conduct thecocultivation may be repeated once to several times. The conditions ofthe each cocultivation may be the same as described above.

By the above-described cocultivation, cytotoxic T cells specific to thepolypeptide are induced and allowed to proliferate. Thus, using theabove-described polypeptide, isolated T cells can be prepared whichselectively bind the complex between the polypeptide and the MHCmolecule.

As described in the Examples below, the genes encoding the polypeptidesof SEQ ID NOs:2, 16, 26, 28 and 39 and SEQ ID NOs:4, 18, 30 and 41 areexpressed specifically in cancer cells and testis of dogs and humans,respectively. Thus, in cancer cells, significantly higher numbers of thepolypeptides of SEQ ID NOs:2, 16, 26, 28 and 39 or SEQ ID NOs:4, 18, 30and 41 exist than in normal cells. When cytotoxic T cells prepared asdescribed above are administered to a living body while a part of thepolypeptides existing in cancer cells are presented by MHC molecules onthe surfaces of the cancer cells, the cytotoxic T cells can damage thecancer cells using the presented polypeptides as markers. Sinceantigen-presenting cells presenting the above-described polypeptides caninduce, and allow proliferation of, cytotoxic T cells specific to thepolypeptides also in vivo, cancer cells can be damaged also byadministering the antigen-presenting cells to a living body. That is,the above-described cytotoxic T cells and the above-describedantigen-presenting cells prepared using the above-described polypeptideare also effective as therapeutic and/or prophylactic agents for acancer(s).

In cases where the above-described isolated antigen-presenting cells orisolated T cells are administered to a living body, these are preferablyprepared by treating antigen presenting cells or T cells collected fromthe patient to be treated with the polypeptide (a) to (c) as describedabove in order to avoid the immune response in the living body thatattacks these cells as foreign bodies.

The therapeutic and/or prophylactic agent for a cancer(s) comprising asan effective ingredient the antigen-presenting cells or T cells ispreferably administered via a parenteral administration route such asintravenous or intraarterial administration. The dose is appropriatelyselected depending on the symptom, the purpose of administration and thelike, and is usually 1 cell to 10,000,000,000,000 cells, preferably1,000,000 cells to 1,000,000,000 cells, which dose is preferablyadministered once per several days to once per several months. Theformulation may be, for example, the cells suspended in physiologicalbuffered saline, and the formulation may be used in combination withanother/other anticancer preparation(s) and/or cytokine(s). Further, oneor more additives well-known in the field of formulation ofpharmaceuticals may also be added.

Also by expression of the polynucleotide encoding the above-describedpolypeptide (a) to (c) in the body of the animal to be treated, antibodyproduction and cytotoxic T cells can be induced in the living body, andan effect comparable to the administration of a polypeptide can beobtained. That is, the immunity-inducing agent of the present inventionmay be one comprising as an effective ingredient a recombinant vectorhaving a polynucleotide encoding the above-described polynucleotide (a)to (c), which recombinant vector is capable of expressing thepolypeptide in a living body. Such a recombinant vector capable ofexpressing an antigenic polypeptide is also called gene vaccine. Thevector used for production of a gene vaccine is not restricted as longas it is a vector capable of expressing a polypeptide in cells of theanimal to be treated (preferably in a mammalian cell), and may be eithera plasmid vector or a virus vector, and any known vector in the field ofgene vaccines may be used. The polynucleotide such as DNA or RNAencoding the above-described polypeptide can be easily prepared, asmentioned above, by a conventional method. Incorporation of thepolynucleotide into a vector can be carried out using a methodwell-known to those skilled in the art.

The administration route of the gene vaccine is preferably a parenteralroute such as intramuscular, subcutaneous, intravenous or intraarterialadministration, and the dose may be appropriately selected depending onthe type of the antigen and the like, and usually about 0.1 μg to 100mg, preferably about 1 μg to 10 mg in terms of the weight of the genevaccine per 1 kg of body weight.

Methods using a virus vector include those wherein a polynucleotideencoding the above-described polypeptide is incorporated into an RNAvirus or DNA virus such as retrovirus, adenovirus, adeno-associatedvirus, herpes virus, vaccinia virus, pox virus, poliovirus or Sindbisvirus, and then the animal to be treated is infected by the resultingvirus. Among these methods, those using retrovirus, adenovirus,adeno-associated virus, vaccinia virus or the like are especiallypreferred.

Other methods include a method wherein an expression plasmid is directlyintramuscularly administered (DNA vaccine method), the liposome method,lipofectin method, microinjection method, calcium phosphate method,electroporation method and the like, and the DNA vaccine method andliposome method are especially preferred.

Methods for actually making the gene encoding the above-describedpolypeptide of the present invention act as a pharmaceutical include thein vivo method wherein the gene is directly introduced into the body,and the ex vivo method wherein a kind of cells are collected from theanimal to be treated, the gene is introduced into the cells ex vivo, andthen the cells are returned to the body (Nikkei Science, 1994, April, p.20-45; The Pharmaceutical Monthly, 1994, Vol. 36, No. 1, p. 23-48;Experimental Medicine, Extra Edition, 1994, Vol. 12, No. 15; andreferences cited in these papers and the like). The in vivo method ismore preferred.

In cases where the gene is administered by the in vivo method, the genemay be administered through an appropriate administration routedepending on the disease to be treated, symptom and so on. It may beadministered, for example, by intravenous, intraarterial, subcutaneous,intramuscular administration or the like. In cases where the gene isadministered by the in vivo method, the gene may be formulated into apreparation such as a solution, and in general, it is formulated into aninjection solution or the like containing the DNA encoding theabove-described peptide of the present invention as an effectiveingredient. A commonly used carrier(s) may be added as required. In thecase of a liposome or membrane fusion liposome (Sendai virus(HVJ)-liposome or the like) containing the DNA, the liposome may beformulated into a liposome preparation such as a suspension, frozenpreparation or centrifugally concentrated frozen preparation.

In the present invention, “the base sequence shown in SEQ ID NO:1”includes not only the base sequence expressly written in SEQ ID NO:1,but also the sequence complementary thereto. Thus, “a polynucleotidehaving the base sequence shown in SEQ ID NO:1” includes asingle-stranded polynucleotide having the base sequence expresslywritten in SEQ ID NO:1, a single-stranded polynucleotide having the basesequence complementary thereto, and a double-stranded polynucleotidecomposed of these single strand polynucleotides. When the polynucleotideencoding the polypeptide used in the present invention is prepared, anyone of these base sequences should be appropriately selected, and thoseskilled in the art can easily carry out the selection.

EXAMPLES

The present invention will now be described more concretely by way ofExamples.

Example A-1 Acquisition of Novel Cancer Antigen Protein by SEREX Method(1) Preparation of cDNA Library

Total RNA was prepared from testis tissue of a healthy dog by the Acidguanidium-Phenol-Chloroform method, and poly(A) RNA was purified usingOligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co.,Ltd.) in accordance with the protocol attached to the kit.

Using the obtained mRNA (5 μg), a dog testis cDNA phage library wassynthesized. Preparation of the cDNA phage library was carried out usingcDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack IIIGold Cloning Kit (manufactured by STRATAGENE) in accordance with theprotocols attached to the kits. The size of the prepared cDNA phagelibrary was 1.3×10⁶ pfu/ml.

(2) Screening of cDNA Library with Serum

Using the dog testis-derived cDNA phage library prepared as describedabove, immunoscreening was carried out. More particularly, host E. colicells (XL1-Blue MRF′) were infected with the library such that 2,340clones should appear on an NZY agarose plate having the size of Φ90×15mm, and cultured at 42° C. for 3 to 4 hours to allow the phage to formplaques. The plate was covered with nitrocellulose membrane (Hybond CExtra: manufactured by GE Healthcare Bio-Science) impregnated with IPTG(isopropyl-β-D-thiogalactoside) at 37° C. for 4 hours to induce andexpress proteins, which were thus transferred to the membrane.Subsequently, the membrane was recovered and soaked in TBS (10 mMTris-HCl, 150 mM NaCl; pH 7.5) containing 0.5% non-fat dry milk,followed by shaking it at 4° C. overnight to suppress non-specificreactions. This filter was allowed to react with 500-fold diluted caninepatient serum at room temperature for 2 to 3 hours.

As the above-described canine patient serum, serum collected from caninepatients suffering from squamous cell carcinoma was used. The serum wasstored at −80° C. and pretreated immediately before use. The method ofthe pretreatment of the serum was as follows. That is, host E. colicells (XL1-Blue MRF′) were infected with λ ZAP Express phage to which noforeign gene was inserted, and then cultured on NZY plate medium at 37°C. overnight. Subsequently, the buffer of 0.2 M NaHCO₃, pH 8.3containing 0.5 M NaCl was added to the plate, and the plate was left tostand at 4° C. for 15 hours, followed by collecting the supernatant asan E. coli/phage extract. Thereafter, the collected E. coli/phageextract was allowed to flow through an NHS column (manufactured by GEHealthcare Bio-Science) to immobilize proteins derived from the E.coli/phage thereon. The serum from the canine patients was allowed toflow through and react with this protein-immobilized column to removeantibodies adsorbed on E. coli and/or the phage. The serum fraction thatpassed through the column was 500-fold diluted with TBS containing 0.5%non-fat dry milk, and the resulting diluent was used as the material forthe immunoscreening.

The membrane on which the thus treated serum and the above-describedfusion protein were blotted was washed 4 times with TBS-T (0.05% Tween20/TBS), and allowed to react with goat anti-dog IgG (Goat anti DogIgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 5000-folddiluted with TBS containing 0.5% non-fat dry milk as a secondaryantibody at room temperature for 1 hour, followed by detection by theenzyme coloring reaction using the NBT/BCIP reaction solution(manufactured by Roche). Colonies at positions where a positive coloringreaction was observed were recovered from the NZY agarose plate havingthe size of Φ90×15 mm, and dissolved in 500 μl of SM buffer (100 mMNaCl, 10 mM MgClSO₄, 50 mM Tris-HCl, 0.01% gelatin; pH 7.5). Thescreening was repeated as a second and third screening in the samemanner as described above until a single coloring reaction-positivecolony was obtained, thereby isolating one positive clone afterscreening of 30,940 phage clones reactive with IgG in the serum.

(3) Homology Search of Isolated Antigen Gene

To subject the single positive clone isolated by the above-describedmethod to a base sequence analysis, an operation of conversion of thephage vector to a plasmid vector was carried out. More particularly, 200μl of a solution prepared to contain a host E. coli (XL1-Blue MRF′) suchthat the absorbance OD₆₀₀ should be 1.0 was mixed with 100 μl of apurified phage solution and further with 1 μl of ExAssist helper phage(manufactured by STRATAGENE), and the reaction was allowed to proceed at37° C. for 15 minutes. To the reaction mixture, 3 ml of LB medium wasadded, and the mixture was cultured at 37° C. for 2.5 to 3 hours,followed by immediate incubation in a water bath at 70° C. for 20minutes. The mixture was then centrifuged at 4° C. at 1000×g for 15minutes, and the supernatant was recovered as a phagemid solution.Subsequently, 200 μl of a solution prepared to contain a phagemid hostE. coli (SOLR) such that the absorbance OD₆₀₀ should be 1.0 was mixedwith 10 μl of a purified phage solution, and the reaction was allowed toproceed at 37° C. for 15 minutes. Thereafter, 50 μl of the reactionmixture was plated on ampicillin (final concentration: 50μg/ml)-containing LB agar medium, and cultured at 37° C. overnight. Asingle colony of transformed SOLR was recovered and cultured inampicillin (final concentration: 50 μg/ml)-containing LB medium at 37°C., followed by purification of plasmid DNA having an insert of interestusing QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).

The purified plasmid was subjected to an analysis of the entire sequenceof the insert by the primer walking method using the T3 primer describedin SEQ ID NO:5 and the T7 primer described in SEQ ID NO:6. By thissequence analysis, the gene sequence described in SEQ ID NO:1 wasobtained. Using the base sequence and the amino acid sequence of thisgene, homology search against known genes was carried out using ahomology search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As aresult, it was revealed that the obtained gene is the gene (AccessionNo. XM_535343) encoding a protein (Accession No. XP_535343) whosefunction is unknown. The human homologous factor of this gene was thegene (Accession No. NM_152660) encoding a protein (Accession No.NP_689873) whose function is also unknown (homology: base sequence, 93%;amino acid sequence, 99%). The base sequence of the human homologousfactor is shown in SEQ ID NO:3, and the amino acid sequence thereof isshown in SEQ ID NO:4.

(4) Analysis of Expression in Each Tissue

The expression of the gene, which was obtained by the above-describedmethod, in normal tissues and various cell lines of dog and human wereinvestigated by the RT-PCR (Reverse Transcription-PCR) method. Thereverse transcription reaction was carried out as follows. That is,total RNA was extracted from 50 to 100 mg of each tissue or 5 to 10×10⁶cells of each cell line using TRIZOL reagent (manufactured byInvitrogen) in accordance with the protocol attached to the kit. Usingthis total RNA, cDNA was synthesized by Superscript First-StrandSynthesis System for RT-PCR (manufactured by Invitrogen) in accordancewith the protocol attached to the kit. As the cDNAs from human normaltissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA(manufactured by Invitrogen), QUICK-Clone cDNA (manufactured byCLONTECH) and Large-Insert cDNA Library (manufactured by CLONTECH) wereused. The PCR reactions were carried out as follows using primers(described in SEQ ID NOs:7 and 8) specific to the obtained canine geneand its human homologous gene. That is, respective reagents and theattached buffer were mixed such that the mixture should contain 0.25 μlof the sample prepared by the reverse transcription reaction, 2 μM eachof the above primers, 0.2 mM each of dNTP and 0.65 U of ExTaq polymerase(manufactured by Takara Shuzo Co., Ltd.) in a total volume of 25 μl, andthe reaction was carried out with 30 cycles of 94° C. for 30 seconds,55° C. for 30 seconds and 72° C. for 1 minute using Thermal Cycler(manufactured by BIO RAD). The gene-specific primers having the basesequences shown in the above-described SEQ ID NOs:7 and 8 were thosewhich amplify the regions of the 87th to 606th bases of the basesequence of SEQ ID NO:1 and the 173rd to 695th bases of the basesequence of SEQ ID NO:3, and can be used for investigation of theexpression of both the canine gene and its human homologous gene. As acontrol for comparison, primers (described in SEQ ID NOs:9 and 10)specific to GAPDH were used simultaneously. As a result, as shown inFIG. 1, strong expression of the obtained canine gene was observed intestis among the normal dog tissues, and on the other hand, strongexpression was observed in the canine breast cancer cell line.Expression of the human homologous gene was confirmed, as is the casewith the canine gene, only in testis among the human normal tissues, butthe expression was detected in brain tumor, leukemia, breast cancer andlung cancer cells among human cancer cell lines. Thus, the humanhomologous gene was also confirmed to be specifically expressed intestis and cancer cells.

In FIG. 1, reference numeral 1 in the ordinate indicates the expressionpattern of the above identified gene, and reference numeral 2 indicatesthe expression pattern of the GAPDH gene as a control for comparison.

Example A-2 Preparation of Novel Cancer Antigen Proteins (1) Preparationof Recombinant Protein

Based on the gene of SEQ ID NO:1 obtained in Example A-1, a recombinantprotein was prepared by the following method. Respective reagents andthe attached buffer were mixed such that the mixture should contain 1 μlof the vector which was prepared from the phagemid solution obtained inExample A-1 and was subjected to the sequence analysis, 0.4 μM each oftwo kinds of primers having NdeI and XhoI restriction sites (describedin SEQ ID NOs:11 and 12), 0.2 mM dNTP and 1.25 U of PrimeSTAR HSpolymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of50 μl, and PCR was carried out with 30 cycles of 98° C. for 10 seconds,55° C. for 15 seconds and 72° C. for 1 minute using Thermal Cycler(manufactured by BIO RAD). The above-described two kinds of primers werethose which amplify the region encoding the entire amino acid sequenceof SEQ ID NO:2. After the PCR, the amplified DNA was subjected toelectrophoresis using 1% agarose gel, and a DNA fragment of about 930 bpwas purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and plasmids were recovered thereafter, followed byconfirming, by sequencing, that the amplified gene fragment matches thesequence of interest. The plasmid that matched the sequence of interestwas treated with restriction enzymes NdeI and XhoI and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET16b (manufactured byNovagen) that had been treated with NdeI and XhoI. Usage of this vectorenables production of a His-tag fusion recombinant protein. E. coli forexpression, BL21 (DE3), was transformed with this plasmid, andexpression of the protein of interest was induced in E. coli with 1 mMIPTG.

On the other hand, based on the gene of SEQ ID NO:3, a recombinantprotein of the human homologous gene was prepared by the followingmethod. Respective reagents and the attached buffer were mixed such thatthe mixture should contain 1 μl of the cDNA prepared in Example A-1whose expression could be confirmed by the RT-PCR method in varioustissues/cells, 0.4 μM each of two kinds of primers having EcoRV andEcoRI restriction sites (described in SEQ ID NOs:13 and 14), 0.2 mM dNTPand 1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co.,Ltd.) in a total volume of 50 μl, and PCR was carried out with 30 cyclesof 98° C. for 10 seconds, 55° C. for 15 seconds and 72° C. for 1 minuteusing Thermal Cycler (manufactured by BIO RAD). The above-described twokinds of primers were those which amplify the region encoding the entireamino acid sequence of SEQ ID NO:4. After the PCR, the amplified DNA wassubjected to electrophoresis using 1% agarose gel, and a DNA fragment ofabout 930 bp was purified using QIAquick Gel Extraction Kit(manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and plasmids were recovered thereafter, followed byconfirming, by sequencing, that the amplified gene fragment matches thesequence of interest. The plasmid that matched the sequence of interestwas treated with restriction enzymes EcoRV and EcoRI and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30a (manufactured byNovagen) that had been treated with EcoRV and EcoRI. Usage of thisvector enables production of a His-tag fusion recombinant protein. E.coli for expression, BL21 (DE3), was transformed with this plasmid, andexpression of the protein of interest was induced in E. coli with 1 mMIPTG.

(2) Purification of Recombinant Protein

The above-obtained recombinant E. coli cells that expressed SEQ ID NO:1and SEQ ID NO:3, respectively, were cultured in 100 μg/mlampicillin-containing LB medium at 37° C. until the absorbance at 600 nmreached about 0.7, and then isopropyl-β-D-1-thiogalactopyranoside wasadded thereto such that its final concentration should be 1 mM, followedby culturing them at 37° C. for 4 hours. Subsequently, the cells werecollected by centrifugation at 4,800 rpm for 10 minutes. The pellet ofthe cells was suspended in phosphate-buffered saline and furthersubjected to centrifugation at 4,800 rpm for 10 minutes to wash thecells.

The cells were suspended in 50 mM Tris-HCl buffer (pH 8.0) and subjectedto sonication on ice. The sonicated solution of E. coli was centrifugedat 6,000 rpm for 20 minutes to obtain the supernatant as the solublefraction and the precipitate as the insoluble fraction.

The insoluble fraction was suspended in 50 mM Tris-HCl buffer (pH 8.0)and centrifuged at 6,000 rpm for 15 minutes. This operation was repeatedtwice and an operation of removal of proteases was carried out.

The residue was suspended in 6M guanidine hydrochloride, 0.15 M sodiumchloride-containing 50 mM Tris-HCl buffer (pH 8.0), and the resultingsuspension was left to stand at 4° C. for 20 hours to denature proteins.Thereafter, the suspension was centrifuged at 6,000 rpm for 30 minutes,and the obtained soluble fraction was placed in a nickel chelate columnprepared by a conventional method (carrier: Chelating Sepharose(trademark) Fast Flow (GE Health Care); column volume: 5 mL;equilibration buffer: 6M guanidine hydrochloride, 0.15 M sodiumchloride-containing 50 mM Tris-HCl buffer (pH 8.0)), followed by leavingit to stand at 4° C. overnight to allow adsorption to thenickel-chelated carrier. The supernatant was recovered by centrifugationof this column carrier at 1,500 rpm for 5 minutes, and the columncarrier was suspended in phosphate-buffered saline, followed byrefilling the column with the resulting suspension.

The fraction that was not adsorbed to the column was washed away with 10column volumes of 0.5 M sodium chloride-containing 0.1 M acetate buffer(pH 4.0), and elution was immediately carried out with 0.5 M sodiumchloride-containing 0.1 M acetate buffer (pH 3.0) to obtain a purifiedfraction, which was used as the material for administration teststhereafter. The proteins of interest in respective eluted fractions wereconfirmed by Coomassie staining carried out according to a conventionalmethod. Among these, the canine protein of interest is shown in FIG. 2.

The buffer contained in the purified preparation obtained by theabove-described method was replaced with a reaction buffer (50 mMTris-HCl, 100 mM NaCl, 5 mM CaCl₂; pH8.0), and cleavage of His tag byFactor Xa protease and purification of the protein of interest werecarried out, using FactorXa Cleavage Capture Kit (manufactured byNovagen), in accordance with the protocols attached to the kit.Subsequently, the buffer contained in 1.2 ml of the purified preparationobtained by the above-described method was replaced with physiologicalphosphate buffer (manufactured by Nissui Pharmaceutical) byultrafiltration using NANOSEP 10K OMEGA (manufactured by PALL), and theresulting solution was filtered aseptically using HT Tuffryn Acrodisc0.22 μm (manufactured by PALL) and used in the following experiments.

Example A-3 Test of Administration of Recombinant Protein toCancer-Bearing Dogs (1) Antitumor Assay

The anti-tumor effect of the two kinds of recombinant proteins whichwere purified as described above was assessed in two individuals ofcancer-bearing dogs having epidermal tumor (2 individuals having mammarygland tumor).

An equal amount of Freund's incomplete adjuvant (manufactured by WakoPure Chemicals) was mixed with 100 μg (0.5 ml) of the recombinantpolypeptides (derived from dog and human), respectively, to prepare twokinds of therapeutic agents for a cancer(s). Each of these agents wasadministered to a regional lymph node in the vicinity of the tumor atotal of 3 times, by carrying out the subsequent administrations 3 daysand 7 days after the first administration. As a result, the tumors witha size of about 25 mm³ and 50 mm³ at the time of administration of thetherapeutic agents for a cancer(s) (derived from dog and human),respectively, were reduced in size to 20 mm³ and 42 mm³, respectively,10 days after the first administration; 13 mm³ and 26 mm³, respectively,20 days after the first administration; and to 5 mm³ and 10 mm³,respectively, 30 days after the first administration.

Further, to a canine patient suffering from malignant melanoma, amixture of 100 μg (0.5 ml) of the above-described polypeptide derivedfrom dog and 0.5 ml of Freund's incomplete adjuvant was administeredintracutaneously at the periphery of the tumor a total of 3 times at thesame intervals as described above. Further, concurrently with therespective administrations, 10 MU of “Intercat” which is a recombinantfeline interferon was administered subcutaneously. As a result, thetumor with a size of about 142 mm³ at the time of administration of thetherapeutic agent for a cancer(s) completely regressed 29 days after thefirst administration.

Further, to a canine patient suffering from nasal adenocarcinoma, amixture of 100 μg (0.5 ml) of the above-described polypeptide derivedfrom dog and 0.5 ml of Freund's incomplete adjuvant was administered inthe same manner as described above a total of 3 times. Further,concurrently with the respective administrations, 100 μg of canineinterleukin 12 was administered subcutaneously. As a result, the tumorwith a size of about 57 mm³ at the time of administration of thetherapeutic agent for a cancer(s) completely regressed 14 days after thefirst administration.

(2) Immune Inducibility Assay

Blood from the canine patient in which the anti-tumor effect wasobtained in the administration test in the above-described (1) wascollected before administration of the therapeutic agent for acancer(s), and 10 days and 30 days after the first administration.Peripheral blood mononuclear cells were isolated according to aconventional method, and by the ELISPOT assay for IFNγ using it, theimmune inducibility of each administered recombinant protein wasassayed.

In a 96-well plate manufactured by Millipore (MultiScreen-IP, MAIPS4510), 100 μL/well of 70% ethanol was placed and the plate was left tostand for 5 minutes, followed by removal of the ethanol by aspiration.The plate was washed with sterile water and 300 μl/well of 200 mM SodiumBicarbonate (pH8.2) was placed therein. After leaving it to stand for 5minutes, Sodium Bicarbonate was removed by aspiration, and then theplate was washed. Subsequently, 0.5 μl/well of anti-canine interferon γmonoclonal antibody (manufactured by R&D, clone 142529, MAB781) mixedwith 200 mM Sodium Bicarbonate was placed in wells, and the plate wasincubated at 37° C. overnight to immobilize the primary antibody. Afterremoval of the primary antibody by aspiration, 300 μL/well of a blockingsolution (1% BSA-5% sucrose-200 mM Sodium Bicarbonate (pH8.2)) was addedto the wells, and the plate was incubated at 4° C. overnight to blockthe plate. After removal of the blocking solution by aspiration, 300μL/well of 10% fetal calf serum-containing RPMI medium (manufactured byInvitrogen) was placed in the wells, and the plate was left to stand for5 minutes, followed by removal of the medium by aspiration.Subsequently, 5×10⁵ cells/well of the canine peripheral bloodmononuclear cells suspended in 10% fetal calf serum-containing RPMImedium were placed in the plate, and 10 μL/well of the canine-derivedpolypeptide or human-derived polypeptide used in each administration wasadded thereto, followed by culturing the cells under the conditions of37° C. and 5% CO₂ for 24 hours, to allow immunocytes that might exist inthe peripheral blood mononuclear cells to produce interferon γ. Afterthe culture, the medium was removed, and the wells were washed 6 timeswith a washing solution (0.1% Tween20-200 mM Sodium Bicarbonate(pH8.2)). In each well, 100 μL of rabbit anti-dog polyclonal antibody1000-fold diluted with the above-described blocking solution was placed,and the plate was incubated at 4° C. overnight. After washing the wells3 times with the above-described washing solution, 100 μL of HRP-labeledanti-rabbit antibody 1000-fold diluted with the above-described blockingsolution was placed in each well, and the reaction was allowed toproceed at 37° C. for 2 hours. After washing the wells 3 times with theabove-described washing solution, the resultant was colored with KonicaImmunostain (manufactured by Konica), and the wells were washed withwater to stop the reaction. Thereafter, the membrane was dried, and thenumber of the appeared spots was counted using KS ELISPOT (manufacturedby Carl Zeiss, Inc.).

As a result, in either canine patient to which the canine polypeptide orthe human polypeptide was administered, peripheral blood mononuclearcells sampled before the administration of the polypeptide showed nospots. On the other hand, in the canine patient to which the caninepolypeptide was administered, peripheral blood mononuclear cells sampled10 days and 30 days after the administration showed 20 and 36 spots,respectively. In the canine patient to which the human polypeptide wasadministered, peripheral blood mononuclear cells sampled 10 days and 30days after the administration showed 24 and 36 spots, respectively.

From the above results, it is confirmed that immunocytes whichspecifically react with the administered recombinant protein and produceinterferon γ were induced in all of the canine patients to which therecombinant protein was administered, and it is thought that theanti-tumor effect described in (1) was exerted by immunoreactions inwhich these immunocytes are mainly involved.

Example B-1 Acquisition of Novel Cancer Antigen Protein by SEREX Method(1) Preparation of cDNA Library

Total RNA was prepared from testis tissue of a healthy dog by the Acidguanidium-Phenol-Chloroform method, and poly(A) RNA was purified usingOligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co.,Ltd.) in accordance with the protocol attached to the kit.

Using the obtained mRNA (5 μg), a dog testis cDNA phage library wassynthesized. Preparation of the cDNA phage library was carried out usingcDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack IIIGold Cloning Kit (manufactured by STRATAGENE) in accordance with theprotocols attached to the kits. The size of the prepared cDNA phagelibrary was 1.3×10⁶ pfu/ml.

(2) Screening of cDNA Library with Serum

Using the dog testis-derived cDNA phage library prepared as describedabove, immunoscreening was carried out. More particularly, host E. colicells (XL1-Blue MRF′) were infected with the library such that 2,340clones should appear on an NZY agarose plate having the size of Φ90×15mm, and cultured at 42° C. for 3 to 4 hours to allow the phage to formplaques. The plate was covered with nitrocellulose membrane (Hybond CExtra: manufactured by GE Healthcare Bio-Science) impregnated with IPTG(isopropyl-β-D-thiogalactoside) at 37° C. for 4 hours to induce andexpress proteins, which were thus transferred to the membrane.Subsequently, the membrane was recovered and soaked in TBS (10 mMTris-HCl, 150 mM NaCl; pH 7.5) containing 0.5% non-fat dry milk,followed by shaking at 4° C. overnight to suppress non-specificreactions. This filter was allowed to react with 500-fold diluted caninepatient serum at room temperature for 2 to 3 hours.

As the above-described canine patient serum, serum collected from caninepatients suffering from tumor proximal to the anus was used. The serumwas stored at −80° C. and pretreated immediately before use. The methodof the pretreatment of the serum was as follows. That is, host E. colicells (XL1-Blue MRF′) were infected with λ ZAP Express phage to which noforeign gene was inserted, and then cultured on NZY plate medium at 37°C. overnight. Subsequently, the buffer of 0.2 M NaHCO₃, pH 8.3containing 0.5 M NaCl was added to the plate, and the plate was left tostand at 4° C. for 15 hours, followed by collecting the supernatant asan E. coli/phage extract. Thereafter, the collected E. coli/phageextract was allowed to flow through an NHS column (manufactured by GEHealthcare Bio-Science) to immobilize proteins derived from the E.coli/phage thereon. The serum from the canine patients was allowed toflow through and react with this protein-immobilized column to removeantibodies adsorbed on E. coli and/or the phage. The serum fraction thatpassed through the column was 500-fold diluted with TBS containing 0.5%non-fat dry milk, and the resulting diluent was used as the material forthe immunoscreening.

The membrane on which the thus treated serum and the above-describedfusion protein were blotted was washed 4 times with TBS-T (0.05% Tween20/TBS), and allowed to react with goat anti-dog IgG (Goat anti DogIgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 5000-folddiluted with TBS containing 0.5% non-fat dry milk as a secondaryantibody at room temperature for 1 hour, followed by detection by theenzyme coloring reaction using the NBT/BCIP reaction solution(manufactured by Roche). Colonies at positions where a positive coloringreaction was observed were recovered from the NZY agarose plate havingthe size of Φ90×15 mm, and dissolved in 500 μl of SM buffer (100 mMNaCl, 10 mM MgClSO₄, 50 mM Tris-HCl, 0.01% gelatin; pH 7.5). Thescreening was repeated as a second and third screening in the samemanner as described above until a single coloring reaction-positivecolony was obtained, thereby isolating one positive clone afterscreening of 30,940 phage clones reactive with IgG in the serum.

(3) Homology Search of Isolated Antigen Gene

To subject the single positive clone isolated by the above-describedmethod to a base sequence analysis, an operation of conversion of thephage vector to a plasmid vector was carried out. More particularly, 200μl of a solution prepared to contain a host E. coli (XL1-Blue MRF′) suchthat the absorbance OD₆₀₀ should be 1.0 was mixed with 100 μl of apurified phage solution and further with 1 μl of ExAssist helper phage(manufactured by STRATAGENE), and the reaction was allowed to proceed at37° C. for 15 minutes. To the reaction mixture, 3 ml of LB medium wasadded, and the mixture was cultured at 37° C. for 2.5 to 3 hours,followed by immediate incubation in a water bath at 70° C. for 20minutes. The mixture was then centrifuged at 4° C. at 1000×g for 15minutes, and the supernatant was recovered as a phagemid solution.Subsequently, 200 μl of a solution prepared to contain a phagemid hostE. coli (SOLR) such that the absorbance OD₆₀₀ should be 1.0 was mixedwith 10 μl of a purified phage solution, and the reaction was allowed toproceed at 37° C. for 15 minutes. Thereafter, 50 μl of the reactionmixture was plated on ampicillin (final concentration: 50μg/ml)-containing LB agar medium, and cultured at 37° C. overnight. Asingle colony of transformed SOLR was recovered and cultured inampicillin (final concentration: 50 μg/ml)-containing LB medium at 37°C., followed by purification of plasmid DNA having an insert of interestusing QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).

The purified plasmid was subjected to an analysis of the entire sequenceof the insert by the primer walking method using the T3 primer describedin SEQ ID NO:5 and the T7 primer described in SEQ ID NO:6. By thissequence analysis, the gene sequence described in SEQ ID NO:15 wasobtained. Using the base sequence and the amino acid sequence of thisgene, homology search against known genes was carried out using ahomology search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As aresult, it was revealed that the obtained gene is the calmegin gene. Thehuman homologous factor of the canine calmegin gene was human calmegin(homology: base sequence, 90%; amino acid sequence, 89%). The basesequence of human calmegin is shown in SEQ ID NO:17, and the amino acidsequence thereof is shown in SEQ ID NO:18.

(4) Analysis of Expression in Each Tissue

The expression of the gene, which was obtained by the above-describedmethod, in normal tissues and various cell lines of dog and human wereinvestigated by the RT-PCR (Reverse Transcription-PCR) method. Thereverse transcription reaction was carried out as follows. That is,total RNA was extracted from 50 to 100 mg of each tissue or 5 to 10×10⁶cells of each cell line using TRIZOL reagent (manufactured byInvitrogen) in accordance with the protocol attached to the kit. Usingthis total RNA, cDNA was synthesized by Superscript First-StrandSynthesis System for RT-PCR (manufactured by Invitrogen) in accordancewith the protocol attached to the kit. As the cDNAs from human normaltissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA(manufactured by Invitrogen), QUICK-Clone cDNA (manufactured byCLONTECH) and Large-Insert cDNA Library (manufactured by CLONTECH) wereused. The PCR reactions were carried out as follows using primers(described in SEQ ID NOs:19 and 20) specific to the obtained gene. Thatis, respective reagents and the attached buffer were mixed such that themixture should contain 0.25 μl of the sample prepared by the reversetranscription reaction, 2 μM each of the above primers, 0.2 mM each ofdNTP and 0.65 U of ExTaq polymerase (manufactured by Takara Shuzo Co.,Ltd.) in a total volume of 25 μl, and the reaction was carried out with30 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for1 minute using Thermal Cycler (manufactured by BIO RAD). Theabove-described gene-specific primers were those which amplify theregions of the 755th to 1318th bases of the base sequence of SEQ IDNO:15 (canine calmegin gene) and the 795th to 1358th bases of the basesequence of SEQ ID NO:17 (human calmegin gene), and can be used forinvestigation of the expression of both the canine calmegin gene and thehuman calmegin gene. As a control for comparison, primers (described inSEQ ID NOs:9 and 10) specific to GAPDH were used simultaneously. As aresult, as shown in FIG. 3, strong expression of the canine calmegingene was observed in testis among the normal dog tissues, and on theother hand, strong expression was observed in canine tumor cell lines.Expression of the human calmegin gene was confirmed, as is the case withthe canine calmegin gene, only in testis among the human normal tissues,but the expression was detected in brain tumor, leukemia and esophaguscancer cells among human cancer cell lines. Thus, the human calmegingene was also confirmed to be specifically expressed in testis andcancer cells.

In FIG. 3, reference numeral 1 in the ordinate indicates the expressionpattern of the calmegin gene, and reference numeral 2 indicates theexpression pattern of the GAPDH gene as a control for comparison.

Example B-2 Preparation of Canine and Human Calmegin Proteins (1)Preparation of Recombinant Protein

Based on the gene of SEQ ID NO:15 obtained in Example B-1, a recombinantprotein was prepared by the following method. Respective reagents andthe attached buffer were mixed such that the mixture should contain 1 μlof the vector that was prepared from the phagemid solution obtained inExample B-1 and was subjected to the sequence analysis, 0.4 μM each oftwo kinds of primers having BamHI and EcoRI restriction sites (describedin SEQ ID NOs:21 and 22), 0.2 mM dNTP and 1.25 U of PrimeSTAR HSpolymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of50 μl, and PCR was carried out with 30 cycles of 98° C. for 10 seconds,55° C. for 15 seconds and 72° C. for 2 minutes using Thermal Cycler(manufactured by BIO RAD). The above-described two kinds of primers werethose which amplify the region encoding the entire amino acid sequenceof SEQ ID NO:16. After the PCR, the amplified DNA was subjected toelectrophoresis using 1% agarose gel, and a DNA fragment of about 1.9kbp was purified using QIAquick Gel Extraction Kit (manufactured byQIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and plasmids were recovered thereafter, followed byconfirming, by sequencing, that the amplified gene fragment matches thesequence of interest. The plasmid that matched the sequence of interestwas treated with restriction enzymes BamHI and EcoRI and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30a (manufactured byNovagen) that had been treated with BamHI and EcoRI. Usage of thisvector enables production of a His-tag fusion recombinant protein. E.coli for expression, BL21 (DE3), was transformed with this plasmid, andexpression of the protein of interest was induced in E. coli with 1 mMIPTG.

On the other hand, based on the gene of SEQ ID NO:17, a recombinantprotein of the human homologous gene was prepared by the followingmethod. Respective reagents and the attached buffer were mixed such thatthe mixture should contain 1 μl of the cDNA prepared in Example B-1whose expression could be confirmed by the RT-PCR method in varioustissues/cells, 0.4 μM each of two kinds of primers having EcoRI and XhoIrestriction sites (described in SEQ ID NOs:23 and 24), 0.2 mM dNTP and1.25 U of PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co.,Ltd.) in a total volume of 50 μl, and PCR was carried out with 30 cyclesof 98° C. for 10 seconds, 55° C. for 15 seconds and 72° C. for 2 minutesusing Thermal Cycler (manufactured by BIO RAD). The above-described twokinds of primers were those which amplify the region encoding the entireamino acid sequence of SEQ ID NO:18. After the PCR, the amplified DNAwas subjected to electrophoresis using 1% agarose gel, and a DNAfragment of about 1.9 kbp was purified using QIAquick Gel Extraction Kit(manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and plasmids were recovered thereafter, followed byconfirming, by sequencing, that the amplified gene fragment matches thesequence of interest. The plasmid that matched the sequence of interestwas treated with restriction enzymes EcoRI and XhoI and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30a (manufactured byNovagen) that had been treated with EcoRI and XhoI. Usage of this vectorenables production of a His-tag fusion recombinant protein. E. coli forexpression, BL21 (DE3), was transformed with this plasmid, andexpression of the protein of interest was induced in E. coli with 1 mMIPTG.

(2) Purification of Recombinant Protein

The above-obtained recombinant E. coli cells that expressed SEQ ID NO:15and SEQ ID NO:17, respectively, were cultured in 30 μg/mlkanamycin-containing LB medium at 37° C. until the absorbance at 600 nmreached about 0.7, and then isopropyl-β-D-1-thiogalactopyranoside wasadded thereto such that its final concentration should be 1 mM, followedby culturing them at 37° C. for 4 hours. Subsequently, the cells werecollected by centrifugation at 4,800 rpm for 10 minutes. The pellet ofthe cells was suspended in phosphate-buffered saline and furthersubjected to centrifugation at 4,800 rpm for 10 minutes to wash thecells.

The obtained pellet of E. coli cells was suspended in 20 mM phosphatebuffer (pH 7.0) and subjected to sonication on ice. The sonicatedsolution of E. coli was centrifuged at 6,000 rpm for 20 minutes toobtain the supernatant as the soluble fraction and the precipitate asthe insoluble fraction.

The soluble fraction was placed in an ion-exchange column (carrier: SPSepharose (trademark) Fast Flow (GE Health Care); column volume: 5 mL;equilibration buffer: 20 mM phosphate buffer (pH 7.0)). The column waswashed with 10 column volumes of 20 mM phosphate buffer (pH 7.0), andelution was carried out with density gradient of salt by 0.3 M-1.0 Msodium chloride-containing 20 mM phosphate buffer (pH 7.0). Six columnvolumes of the eluted fraction was collected in each elution step.

Among these eluted fractions, the 1st to 6th fractions eluted with 0.3 Msodium chloride-containing 20 mM phosphate buffer (pH 7.0) and the 1stfraction eluted with 1.0 M sodium chloride-containing 20 mM phosphatebuffer (pH 7.0) were combined, and the resulting solution was subjectedto additional purification by a secondary column.

For the secondary column, a column carrier Bio gel HT Type II (BioRad)was used. The column volume was 5 mL. The column was equilibrated with10 column volumes of 0.3 M sodium chloride-containing 20 mM phosphatebuffer (pH 7.0), and the above-described eluted fractions were placed inthe column. The fractions that were not adsorbed to the column waswashed away with 10 column volumes of 0.3 M sodium chloride-containing20 mM phosphate buffer (pH 7.0) and 0.1 M phosphate buffer (pH 7.0), andelution was carried out with 0.2 M phosphate buffer (pH 7.0) to obtain apurified fraction, which was used as the material for administrationtests thereafter. The proteins of interest in the eluted fractions wereconfirmed by Coomassie staining carried out according to a conventionalmethod. Among these, the canine calmegin protein is shown in FIG. 4.

To 1 ml of a reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl₂; pH7.4), 200 μl of the purified preparation obtained by the above-describedmethod was aliquoted, and 2 μl of enterokinase (manufactured by Novagen)was then added thereto, followed by leaving it to stand at roomtemperature overnight to cleave His tag. The resulting product waspurified using Enterokinase Cleavage Capture Kit (manufactured byNovagen) in accordance with the protocol attached to the kit.Subsequently, the buffer contained in 1.2 ml of the purified preparationobtained by the above-described method was replaced with physiologicalphosphate buffer (manufactured by Nissui Pharmaceutical) byultrafiltration using NANOSEP 10K OMEGA (manufactured by PALL), and theresulting solution was filtered aseptically using HT Tuffryn Acrodisc0.22 μm (manufactured by PALL) and used in the following experiments.

Example B-3 Test of Administration of Recombinant Protein toCancer-Bearing Dogs (1) Antitumor Assay

The anti-tumor effect of the two kinds of recombinant proteins whichwere purified as described above was assessed in two individuals ofcancer-bearing dogs having epidermal tumor (2 individuals having mammarygland tumor).

An equal amount of Freund's incomplete adjuvant (manufactured by WakoPure Chemicals) was mixed with 100 μg (0.5 ml) of the recombinant caninecalmegin and human calmegin proteins, respectively, to preparetherapeutic agents for a cancer(s). Each of these agents wasadministered to a regional lymph node in the vicinity of the tumor atotal of 3 times, by carrying out the subsequent administrations 3 daysand 7 days after the first administration. As a result, the tumors witha size of about 45 mm³ and 78 mm³, respectively, at the time ofadministration of the therapeutice agents were reduced to 27 mm³ and 46mm³, respectively, 10 days after the first administration; 15 mm³ and 26mm³, respectively, 20 days after the first administration; and to 7 mm³and 15 mm³, respectively, 30 days after the first administration.

Further, to a canine patient suffering from malignant melanoma, amixture of 100 μg (0.5 ml) of the above-described canine calmeginprotein and 0.5 ml of Freund's incomplete adjuvant was administered atotal of 3 times in the same manner as described above. Further,concurrently with the respective administrations, 100 μg of canineinterleukin 12 was administered subcutaneously. As a result, the tumorwith a size of about 38 mm³ at the time of administration of thetherapeutic agent completely regressed 21 days after the firstadministration of the therapeutic agent.

(2) Immune Inducibility Assay

Blood from the canine patient in which the anti-tumor effect wasobtained in the administration test in the above-described (1) wascollected before administration of the therapeutic agent for a cancer(s)and 10 days and 30 days after the first administration. Peripheral bloodmononuclear cells were isolated according to a conventional method, andby the ELISPOT assay for IFNγ using it, the immune inducibility of eachadministered recombinant protein was assayed.

In a 96-well plate manufactured by Millipore (MultiScreen-IP, MAIPS4510), 100 μL/well of 70% ethanol was placed and the plate was left tostand for 5 minutes, followed by removal of the ethanol by aspiration.The plate was washed with sterile water and 300 μl/well of 200 mM SodiumBicarbonate (pH8.2) was placed therein. After leaving it to stand for 5minutes, Sodium Bicarbonate was removed by aspiration, and then theplate was washed. Subsequently, 0.5 μg/well of anti-canine interferon γmonoclonal antibody (manufactured by R&D, clone 142529, MAB781) mixedwith 200 mM Sodium Bicarbonate was placed in wells, and the plate wasincubated at 37° C. overnight to immobilize the primary antibody. Afterremoval of the primary antibody by aspiration, 300 μL/well of a blockingsolution (1% BSA-5% sucrose-200 mM Sodium Bicarbonate (pH8.2)) was addedto the wells, and the plate was incubated at 4° C. overnight to blockthe plate. After removal of the blocking solution by aspiration, 300μL/well of 10% fetal calf serum-containing RPMI medium (manufactured byInvitrogen) was placed in the wells, and the plate was left to stand for5 minutes, followed by removal of the medium by aspiration.Subsequently, 5×10⁵ cells/well of the canine peripheral bloodmononuclear cells suspended in 10% fetal calf serum-containing RPMImedium were placed in the plate, and 10 μL/well of the canine calmeginor human calmegin protein used in each administration was added thereto,followed by culturing the cells under the conditions of 37° C. and 5%CO₂ for 24 hours, to allow immunocytes that might exist in theperipheral blood mononuclear cells to produce interferon γ. After theculture, the medium was removed, and the wells were washed 6 times witha washing solution (0.1% Tween20-200 mM Sodium Bicarbonate (pH8.2)). Ineach well, 100 μL of rabbit anti-dog polyclonal antibody 1000-folddiluted with the above-described blocking solution was placed, and theresulting mixture was incubated at 4° C. overnight. After washing thewells 3 times with the above-described washing solution, 100 μL ofHRP-labeled anti-rabbit antibody 1000-fold diluted with theabove-described blocking solution was placed in each well, and thereaction was allowed to proceed at 37° C. for 2 hours. After washing thewells 3 times with the above-described washing solution, the resultantwas colored with Konica Immunostain (manufactured by Konica), and thewells were washed with water to stop the reaction. Thereafter, themembrane was dried, and image processing of the wells was carried out,followed by counting the number of spot-forming cells (SFC) using KSELISPOT compact system (Carl Zeiss, Inc., Germany).

As a result, in either canine patient to which canine calmegin or humancalmegin was administered, peripheral blood mononuclear cells sampledbefore the administration showed no spots. On the other hand, in thecanine patient to which canine calmegin was administered, peripheralblood mononuclear cells sampled 10 days and 30 days after theadministration showed 15 and 45 spots, respectively. In the caninepatient to which human calmegin was administered, peripheral bloodmononuclear cells sampled 10 days and 30 days after the administrationshowed 12 and 39 spots, respectively.

From the above results, it is confirmed that immunocytes whichspecifically react with the administered recombinant protein and produceinterferon γ were induced in all of the canine patients to which therecombinant protein was administered, and it is thought that theanti-tumor effect described in (1) was exerted by immunoreactions inwhich these immunocytes are mainly involved.

Example C-1 Acquisition of Novel Cancer Antigen Protein by SEREX Method(1) Preparation of cDNA Library

Total RNA was prepared from testis tissue of a healthy dog by the Acidguanidium-Phenol-Chloroform method, and poly(A) RNA was purified usingOligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co.,Ltd.) in accordance with the protocol attached to the kit.

Using the obtained mRNA (5 μg), a dog testis cDNA phage library wassynthesized. Preparation of the cDNA phage library was carried out usingcDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack IIIGold Cloning Kit (manufactured by STRATAGENE) in accordance with theprotocols attached to the kits. The size of the prepared cDNA phagelibrary was 1.3×10⁶ pfu/ml.

(2) Screening of cDNA Library with Serum

Using the dog testis-derived cDNA phage library prepared as describedabove, immunoscreening was carried out. More particularly, host E. colicells (XL1-Blue MRF′) were infected with the library such that 2,340clones should appear on an NZY agarose plate having the size of Φ90×15mm, and cultured at 42° C. for 3 to 4 hours to allow the phage to formplaques. The plate was covered with nitrocellulose membrane (Hybond CExtra: manufactured by GE Healthcare Bio-Science) impregnated with IPTG(isopropyl-β-D-thiogalactoside) at 37° C. for 4 hours to induce andexpress proteins, which were thus transferred to the membrane.Subsequently, the membrane was recovered and soaked in TBS (10 mMTris-HCl, 150 mM NaCl; pH 7.5) containing 0.5% non-fat dry milk,followed by shaking it at 4° C. overnight to suppress non-specificreactions. This filter was allowed to react with 500-fold diluted caninepatient serum at room temperature for 2 to 3 hours.

As the above-described canine patient serum, serum collected from caninepatients suffering from breast cancer was used. The serum was stored at−80° C. and pretreated immediately before use. The method of thepretreatment of the serum was as follows. That is, host E. coli cells(XL1-Blue MRF′) were infected with λ ZAP Express phage to which noforeign gene was inserted, and then cultured on NZY plate medium at 37°C. overnight. Subsequently, the buffer of 0.2 M NaHCO₃, pH 8.3containing 0.5 M NaCl was added to the plate, and the plate was left tostand at 4° C. for 15 hours, followed by collecting the supernatant asan E. coli/phage extract. Thereafter, the collected E. coli/phageextract was allowed to flow through an NHS column (manufactured by GEHealthcare Bio-Science) to immobilize proteins derived from the E.coli/phage thereon. The serum from the canine patients was allowed toflow through and react with this protein-immobilized column to removeantibodies adsorbed on E. coli and/or the phage. The serum fraction thatpassed through the column was 500-fold diluted with TBS containing 0.5%non-fat dry milk, and the resulting diluent was used as the material forthe immunoscreening.

The membrane on which the thus treated serum and the above-describedfusion protein were blotted was washed 4 times with TBS-T (0.05% Tween20/TBS), and allowed to react with goat anti-dog IgG (Goat anti DogIgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 5,000-folddiluted with TBS containing 0.5% non-fat dry milk as a secondaryantibody at room temperature for 1 hour, followed by detection by theenzyme coloring reaction using the NBT/BCIP reaction solution(manufactured by Roche). Colonies at positions where a positive coloringreaction was observed were recovered from the NZY agarose plate havingthe size of Φ90×15 mm, and dissolved in 500 μl of SM buffer (100 mMNaCl, 10 mM MgClSO₄, 50 mM Tris-HCl, 0.01% gelatin; pH 7.5). Thescreening was repeated as a second and third screening in the samemanner as described above until a single coloring reaction-positivecolony was obtained, thereby isolating one positive clone afterscreening of 30,940 phage clones reactive with IgG in the serum.

(3) Homology Search of Isolated Antigen Gene

To subject the single positive clone isolated by the above-describedmethod to a base sequence analysis, an operation of conversion of thephage vector to a plasmid vector was carried out. More particularly, 200μl of a solution prepared to contain a host E. coli (XL1-Blue MRF′) suchthat the absorbance OD₆₀₀ should be 1.0 was mixed with 100 μl of apurified phage solution and further with 1 μl of ExAssist helper phage(manufactured by STRATAGENE), and the reaction was allowed to proceed at37° C. for 15 minutes. To the reaction mixture, 3 ml of LB medium wasadded, and the mixture was cultured at 37° C. for 2.5 to 3 hours,followed by immediate incubation in a water bath at 70° C. for 20minutes. The mixture was then centrifuged at 4° C. at 1000×g for 15minutes, and the supernatant was recovered as a phagemid solution.Subsequently, 200 μl of a solution prepared to contain a phagemid hostE. coli (SOLR) such that the absorbance OD₆₀₀ should be 1.0 was mixedwith 10 μl of a purified phage solution, and the reaction was allowed toproceed at 37° C. for 15 minutes. Thereafter, 50 μl of the reactionmixture was plated on ampicillin (final concentration: 50μg/ml)-containing LB agar medium, and cultured at 37° C. overnight. Asingle colony of transformed SOLR was recovered and cultured inampicillin (final concentration: 50 μg/ml)-containing LB medium at 37°C., followed by purification of plasmid DNA having an insert of interestusing QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).

The purified plasmid was subjected to an analysis of the entire sequenceof the insert by the primer walking method using the T3 primer describedin SEQ ID NO:5 and the T7 primer described in SEQ ID NO:6. By thissequence analysis, the gene sequence described in SEQ ID NO:25 wasobtained. Using the base sequence and the amino acid sequence of thisgene, homology search against known genes was carried out using ahomology search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As aresult, it was revealed that the obtained gene has 99% homology (whichwas calculated only in the overlapping region) to the CEP gene describedin SEQ ID NO:27 in terms of base sequence and amino acid sequence, sothat the gene was judged as the CEP gene. The human homologous factor ofthe canine CEP was human CEP (homology to the CEP gene described in SEQID NO:25: base sequence, 87%; amino acid sequence, 84%). The basesequence of human CEP is shown in SEQ ID NO:29, and the amino acidsequence thereof is shown in SEQ ID NO:30.

(4) Analysis of Expression in Each Tissue

The expression of the gene, which was obtained by the above-describedmethod, in normal tissues and various cell lines of dog and human wereinvestigated by the RT-PCR (Reverse Transcription-PCR) method. Thereverse transcription reaction was carried out as follows. That is,total RNA was extracted from 50 to 100 mg of each tissue or 5 to 10×10⁶cells of each cell line using TRIZOL reagent (manufactured byInvitrogen) in accordance with the protocol attached to the kit. Usingthis total RNA, cDNA was synthesized by Superscript First-StrandSynthesis System for RT-PCR (manufactured by Invitrogen) in accordancewith the protocol attached to the kit. As the cDNAs from human normaltissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA(manufactured by Invitrogen), QUICK-Clone cDNA (manufactured byCLONTECH) and Large-Insert cDNA Library (manufactured by CLONTECH) wereused. The PCR reactions were carried out as follows using primers(described in SEQ ID NOs:31 and 32) specific to the obtained gene. Thatis, respective reagents and the attached buffer were mixed such that themixture should contain 0.25 μl of the sample prepared by the reversetranscription reaction, 2 μM each of the above primers, 0.2 mM each ofdNTP and 0.65 U of ExTaq polymerase (manufactured by Takara Shuzo Co.,Ltd.) in a total volume of 25 μl, and the reaction was carried out with30 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for30 seconds using Thermal Cycler (manufactured by BIO RAD). Theabove-described gene-specific primers were those which amplify theregions of the 4582nd to 5124th bases of the base sequences of SEQ IDNOs:25 and 27 (canine CEP gene) and the 4610th to 5152nd bases of thebase sequence of SEQ ID NO:29 (human CEP gene), and can be used forinvestigation of the expression of both the canine CEP gene and thehuman CEP gene. As a control for comparison, primers (described in SEQID NOs:9 and 10) specific to GAPDH were used simultaneously. As aresult, as shown in FIG. 5, strong expression of the canine CEP gene wasobserved in testis among the normal dog tissues, and on the other hand,strong expression was observed in the canine breast cancer cell line.Expression of the human CEP gene was confirmed, as is the case with thecanine CEP gene, only in testis among the human normal tissues, but theexpression was detected in brain tumor, leukemia and esophagus cancercells among human cancer cell lines, and especially, strong expressionwas observed in the leukemia cell line. Thus, the human CEP gene wasalso confirmed to be specifically expressed in testis and cancer cells.

In FIG. 5, reference numeral 1 in the ordinate indicates the expressionpattern of the CEP gene, and reference numeral 2 indicates theexpression pattern of the GAPDH gene as a control for comparison.

Example C-2 Preparation of Canine and Human CEPs (1) Preparation ofRecombinant Protein

Based on the gene of SEQ ID NO:25 obtained in Example C-1, a recombinantprotein was prepared by the following method. Respective reagents andthe attached buffer were mixed such that the mixture should contain 1 μlof the vector that was prepared from the phagemid solution obtained inExample C-1 and was subjected to the sequence analysis, 0.4 μM each oftwo kinds of primers having BamHI and SalI restriction sites (describedin SEQ ID NOs:33 and 34), 0.2 mM dNTP and 1.25 U of PrimeSTAR HSpolymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volume of50 μl, and PCR was carried out with 30 cycles of 98° C. for 10 seconds,55° C. for 5 seconds and 72° C. for 7 minutes using Thermal Cycler(manufactured by BIO RAD). The above-described two kinds of primers werethose which amplify the region encoding the entire amino acid sequenceof SEQ ID NO:26. After the PCR, the amplified DNA was subjected toelectrophoresis using 1% agarose gel, and a DNA fragment of about 7.0kbp was purified using QIAquick Gel Extraction Kit (manufactured byQIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and plasmids were recovered thereafter, followed byconfirming, by sequencing, that the amplified gene fragment matches thesequence of interest. The plasmid that matched the sequence of interestwas treated with restriction enzymes BamHI and SalI and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30a (manufactured byNovagen) that had been treated with BamHI and SalI. Usage of this vectorenables production of a His-tag fusion recombinant protein. E. coli forexpression, BL21 (DE3), was transformed with this plasmid, andexpression of the protein of interest was induced in E. coli with 1 mMIPTG. In the same manner, based on the gene of SEQ ID NO:27, using thecanine testis cDNA as a template and two kinds of primers having BamHIand SalI restriction sites (SEQ ID NOs:33 and 35), a recombinant proteinof the registered canine CEP gene was prepared. The above-described twokinds of primers were those which amplify the region of about 7.8 kbpencoding the entire amino acid sequence of SEQ ID NO:28.

Further, based on the gene of SEQ ID NO:29, a recombinant protein of thehuman homologous gene was prepared by the following method. Respectivereagents and the attached buffer were mixed such that the mixture shouldcontain 1 μl of the cDNA prepared in Example C-1 whose expression couldbe confirmed by the RT-PCR method in various tissues/cells, 0.4 μM eachof two kinds of primers having BamHI and SalI restriction sites(described in SEQ ID NOs:36 and 37), 0.2 mM dNTP and 1.25 U of PrimeSTARHS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volumeof 50 μl, and PCR was carried out with 30 cycles of 98° C. for 10seconds, 55° C. for 5 seconds and 72° C. for 7 minutes using ThermalCycler (manufactured by BIO RAD). The above-described two kinds ofprimers were those which amplify the region encoding the entire aminoacid sequence of SEQ ID NO:30. After the PCR, the amplified DNA wassubjected to electrophoresis using 1% agarose gel, and a DNA fragment ofabout 7.0 kbp was purified using QIAquick Gel Extraction Kit(manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and plasmids were recovered thereafter, followed byconfirming, by sequencing, that the amplified gene fragment matches thesequence of interest. The plasmid that matched the sequence of interestwas treated with restriction enzymes BamHI and SalI and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30a (manufactured byNovagen) that had been treated with BamHI and SalI. Usage of this vectorenables production of a His-tag fusion recombinant protein. E. coli forexpression, BL21 (DE3), was transformed with this plasmid, andexpression of the protein of interest was induced in E. coli with 1 mMIPTG.

(2) Purification of Recombinant Protein

The above-obtained recombinant E. coli cells that expressed SEQ IDNO:25, SEQ ID NO:27, and SEQ ID NO:29, respectively, were cultured in 30μg/ml kanamycin-containing LB medium at 37° C. until the absorbance at600 nm reached about 0.7, and then isopropyl-β-D-1-thiogalactopyranosidewas added thereto such that its final concentration should be 1 mM,followed by culturing them at 30° C. for 20 hours. Subsequently, thecells were collected by centrifugation at 4,800 rpm for 10 minutes. Thepellet of the cells was suspended in phosphate-buffered saline andfurther subjected to centrifugation at 4,800 rpm for 10 minutes to washthe cells.

The cells were suspended in phosphate-buffered saline and subjected tosonication on ice. The sonicated solution of E. coli was centrifuged at7000 rpm for 20 minutes to obtain the supernatant as the solublefraction and the precipitate as the insoluble fraction. The insolublefraction was suspended in 4% Triton X-100 solution and the resultingsuspension was centrifuged at 7000 rpm for 20 minutes. This operationwas repeated twice and an operation of removal of proteases was carriedout. The residue was suspended in 8 M urea-containing 10 mM Tris-HCl,100 mM phosphate buffer (hereinafter referred to as 8 M urea solution)and a protease inhibitor cocktail solution, and the resulting suspensionwas left to stand at 4° C. for 20 hours to denature proteins.

Thereafter, the suspension was centrifuged at 7,000 rpm for 20 minutes,and the obtained soluble fraction was placed in a nickel chelate columnprepared by a conventional method (carrier: Chelating Sepharose(trademark) Fast Flow (GE Health Care); column volume: 5 mL;equilibration buffer: 8M urea solution), followed by leaving it to standat 4° C. overnight. The supernatant was recovered by centrifugation ofthis column carrier at 1,500 rpm for 5 minutes, and the column carrierwas suspended in phosphate-buffered saline, followed by refilling thecolumn with the resulting suspension. The fraction that was not adsorbedto the column was washed away with 5 column volumes of 8 M ureasolution, 10 column volumes of 0.5 M sodium chloride-containing 0.1 Macetate buffer (pH 5.0) and 10 mM imidazole-containing 20 mM phosphatebuffer (pH 8.0), and elution was immediately carried out with afive-step density gradient of 100 mM-500 mM imidazole to obtain apurified fraction, which was used as the material for administrationtests thereafter. The proteins of interest in respective elutedfractions were confirmed by Coomassie staining carried out according toa conventional method. Among these, the recombinant canine CEP describedin SEQ ID NO:26 is shown in FIG. 6.

To 1 ml of a reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl₂; pH7.4), 200 μl of the purified preparation obtained by the above-describedmethod was aliquoted, and 2 μl of enterokinase (manufactured by Novagen)was then added thereto, followed by leaving it to stand at roomtemperature overnight to cleave His tag. The resulting product waspurified using Enterokinase Cleavage Capture Kit (manufactured byNovagen) in accordance with the protocol attached to the kit.Subsequently, the buffer contained in 1.2 ml of the purified preparationobtained by the above-described method was replaced with physiologicalphosphate buffer (manufactured by Nissui Pharmaceutical) byultrafiltration using NANOSEP 10K OMEGA (manufactured by PALL), and theresulting solution was filtered aseptically using HT Tuffryn Acrodisc0.22 μm (manufactured by PALL) and used in the following experiments.

Example C-3 Test of Administration of Recombinant Protein toCancer-Bearing Dogs (1) Antitumor Assay

The anti-tumor effect of the two kinds of recombinant proteins whichwere purified as described above was assessed in two individuals ofcancer-bearing dogs having epidermal tumor (2 individuals havingperianal adenoma).

An equal amount of Freund's incomplete adjuvant (manufactured by WakoPure Chemicals) was mixed with 100 μg (0.5 ml) each of the recombinantcanine CEP described in SEQ ID NO:26 and human CEP purified as describedabove to prepare therapeutic agents for a cancer(s). Each of theseagents was administered to a regional lymph node in the vicinity of thetumor a total of 3 times, by carrying out the subsequent administrations3 days and 7 days after the first administration. As a result, thetumors with a size of about 87 mm³ and 69 mm³ at the time ofadministration of the therapeutic agents, respectively, were reduced to69 mm³ and 56 mm³, respectively, 10 days after the first administration;24 mm³ and 31 mm³, respectively, 20 days after the first administration;and to 10 mm³ and 8 mm³, respectively, 30 days after the firstadministration of the therapeutic agent.

Further, to a canine patient suffering from mammary adenocarcinoma, amixture of 100 μg (0.5 ml) of the canine CEP protein described in SEQ IDNO:26 with 0.5 ml of Freund's incomplete adjuvant was administered atotal of 3 times in the same manner as described above. Further,concurrently with the respective administrations, 10 MU of “Intercat”which is a recombinant feline interferon was administeredsubcutaneously. As a result, the tumor with a size of about 126 mm³ atthe time of administration of the therapeutic agent completely regressed26 days after the first administration of the therapeutic agent.Similarly, in the case where the canine CEP described in SEQ ID NO:28was used, an anti-tumor effect was also observed in a cancer-bearingdog.

Further, to a canine patient of mastocytoma, a mixture of 100 μg (0.5ml) of the canine CEP protein described in SEQ ID NO:26 with 0.5 ml ofFreund's incomplete adjuvant was administered a total of 3 times in thesame manner as described above. Further, concurrently with therespective administrations, 100 μg of canine interleukin-12 wassubcutaneously administered. As a result, the tumor with a size of about83 mm³ at the time of administration of the therapeutic agent completelyregressed 18 days after the first administration of the therapeuticagent.

(2) Immune Inducibility Assay

Blood from the canine patient suffering from perianal adenoma in whichthe anti-tumor effect was obtained in the administration test in theabove-described (1) was collected before administration of thetherapeutic agent for a cancer(s) and 10 days and 30 days after thefirst administration. Peripheral blood mononuclear cells were isolatedaccording to a conventional method, and by the ELISPOT assay for IFNγusing it, the immune inducibility of each administered protein wasassayed.

In a 96-well plate manufactured by Millipore (MultiScreen-IP, MAIPS4510), 100 μL/well of 70% ethanol was placed and the plate was left tostand for 5 minutes, followed by removal of the ethanol by aspiration.The plate was washed with sterile water and 300 μl/well of 200 mM SodiumBicarbonate (pH8.2) was placed therein. After leaving it to stand for 5minutes, Sodium Bicarbonate was removed by aspiration, and then theplate was washed. Subsequently, 0.5 μl/well of anti-canine interferon γmonoclonal antibody (manufactured by R&D, clone 142529, MAB781) mixedwith 200 mM Sodium Bicarbonate was placed in wells, and the plate wasincubated at 37° C. overnight to immobilize the primary antibody. Afterremoval of the primary antibody by aspiration, 300 μL/well of a blockingsolution (1% BSA-5% sucrose-200 mM Sodium Bicarbonate (pH8.2)) was addedto the wells, and the plate was incubated at 4° C. overnight to blockthe plate. After removal of the blocking solution by aspiration, 300μL/well of 10% fetal calf serum-containing RPMI medium (manufactured byInvitrogen) was placed in the wells, and the plate was left to stand for5 minutes, followed by removal of the medium by aspiration.Subsequently, 5×10⁵ cells/well of the canine peripheral bloodmononuclear cells suspended in 10% fetal calf serum-containing RPMImedium were placed in the plate, and 10 μL/well of the canine CEPdescribed in SEQ ID NO:26 or the human CEP used in each administrationwas added thereto, followed by culturing the cells under the conditionsof 37° C. and 5% CO₂ for 24 hours, to allow immunocytes that might existin the peripheral blood mononuclear cells to produce interferon γ. Afterthe culture, the medium was removed, and the wells were washed 6 timeswith a washing solution (0.1% Tween20-200 mM Sodium Bicarbonate(pH8.2)). In each well, 100 μL of rabbit anti-canine polyclonal antibody1000-fold diluted with the above-described blocking solution was placed,and the plate was incubated at 4° C. overnight. After washing the wells3 times with the above-described washing solution, 100 μL of HRP-labeledanti-rabbit antibody 1000-fold diluted with the above-described blockingsolution was placed in each well, and the reaction was allowed toproceed at 37° C. for 2 hours. After washing the wells 3 times with theabove-described washing solution, the resultant was colored with KonicaImmunostain (manufactured by Konica), and the wells were washed withwater to stop the reaction. Thereafter, the membrane was dried, andimage processing of the wells was carried out, followed by counting thenumber of spot-forming cells (SFC) using KS ELISPOT compact system (CarlZeiss, Inc., Germany).

As a result, in either canine patient to which the canine CEP describedin SEQ ID NO: 26 or the human CEP was administered, peripheral bloodmononuclear cells sampled before the administration showed no spots. Onthe other hand, in the canine patient to which the canine CEP wasadministered, peripheral blood mononuclear cells sampled 10 days and 30days after the administration showed 23 and 52 spots, respectively. Inthe canine patient to which the human CEP was administered, peripheralblood mononuclear cells sampled 10 days and 30 days after theadministration showed 19 and 49 spots, respectively.

From the above results, it is confirmed that immunocytes whichspecifically react with the administered recombinant protein and produceinterferon γ were induced in all of the canine patients to which therecombinant protein was administered, and it is thought that theanti-tumor effect described in (1) was exerted by immunoreactions inwhich these immunocytes are mainly involved.

Example D-1 Acquisition of Novel Cancer Antigen Protein by SEREX Method(1) Preparation of cDNA Library

Total RNA was prepared from testis tissue of a healthy dog by the Acidguanidium-Phenol-Chloroform method, and poly(A) RNA was purified usingOligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co.,Ltd.) in accordance with the protocol attached to the kit.

Using the obtained mRNA (5 μg), a dog testis cDNA phage library wassynthesized. Preparation of the cDNA phage library was carried out usingcDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA Gigapack IIIGold Cloning Kit (manufactured by STRATAGENE) in accordance with theprotocols attached to the kits. The size of the prepared cDNA phagelibrary was 1.3×10⁶ pfu/ml.

(2) Screening of cDNA Library with Serum

Using the dog testis-derived cDNA phage library prepared as describedabove, immunoscreening was carried out. More particularly, host E. colicells (XL1-Blue MRF′) were infected with the library such that 2,340clones should appear on an NZY agarose plate having the size of Φ90×15mm, and cultured at 42° C. for 3 to 4 hours to allow the phage to formplaques. The plate was covered with nitrocellulose membrane (Hybond CExtra: manufactured by GE Healthcare Bio-Science) impregnated with IPTG(isopropyl-β-D-thiogalactoside) at 37° C. for 4 hours to induce andexpress proteins, which were thus transferred to the membrane.Subsequently, the membrane was recovered and soaked in TBS (10 mMTris-HCl, 150 mM NaCl; pH 7.5) containing 0.5% non-fat dry milk,followed by shaking at 4° C. overnight to suppress non-specificreactions. This filter was allowed to react with 500-fold diluted caninepatient serum at room temperature for 2 to 3 hours.

As the above-described canine patient serum, serum collected from caninepatients suffering from breast cancer was used. The serum was stored at−80° C. and pretreated immediately before use. The method of thepretreatment of the serum was as follows. That is, host E. coli cells(XL1-Blue MRF′) were infected with λ ZAP Express phage to which noforeign gene was inserted, and then cultured on NZY plate medium at 37°C. overnight. Subsequently, the buffer of 0.2 M NaHCO₃, pH 8.3containing 0.5 M NaCl was added to the plate, and the plate was left tostand at 4° C. for 15 hours, followed by collecting the supernatant asan E. coli/phage extract. Thereafter, the collected E. coli/phageextract was allowed to flow through an NHS column (manufactured by GEHealthcare Bio-Science) to immobilize proteins derived from the E.coli/phage thereon. The serum from the canine patients was allowed toflow through and react with this protein-immobilized column to removeantibodies adsorbed on E. coli and/or the phage. The serum fraction thatpassed through the column was 500-fold diluted with TBS containing 0.5%non-fat dry milk, and the resulting diluent was used as the material forthe immunoscreening.

The membrane on which the thus treated serum and the above-describedfusion protein were blotted was washed 4 times with TBS-T (0.05% Tween20/TBS), and allowed to react with goat anti-dog IgG (Goat anti DogIgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 5,000-folddiluted with TBS containing 0.5% non-fat dry milk as a secondaryantibody at room temperature for 1 hour, followed by detection by theenzyme coloring reaction using the NBT/BCIP reaction solution(manufactured by Roche). Colonies at positions where a positive coloringreaction was observed were recovered from the NZY agarose plate havingthe size of Φ90×15 mm, and dissolved in 500 μl of SM buffer (100 mMNaCl, 10 mM MgClSO₄, 50 mM Tris-HCl, 0.01% gelatin; pH 7.5). Thescreening was repeated as a second and third screening in the samemanner as described above until a single coloring reaction-positivecolony was obtained, thereby isolating one positive clone afterscreening of 30,940 phage clones reactive with IgG in the serum.

(3) Homology Search of Isolated Antigen Gene

To subject the single positive clone isolated by the above-describedmethod to a base sequence analysis, an operation of conversion of thephage vector to a plasmid vector was carried out. More particularly, 200μl of a solution prepared to contain a host E. coli (XL1-Blue MRF′) suchthat the absorbance OD₆₀₀ should be 1.0 was mixed with 100 μl of apurified phage solution and further with 1 μl of ExAssist helper phage(manufactured by STRATAGENE), and the reaction was allowed to proceed at37° C. for 15 minutes. To the reaction mixture, 3 ml of LB medium wasadded, and the mixture was cultured at 37° C. for 2.5 to 3 hours,followed by immediate incubation in a water bath at 70° C. for 20minutes. The mixture was then centrifuged at 4° C. at 1000×g for 15minutes, and the supernatant was recovered as a phagemid solution.Subsequently, 200 μl of a solution prepared to contain a phagemid hostE. coli (SOLR) such that the absorbance OD₆₀₀ should be 1.0 was mixedwith 10 μl of a purified phage solution, and the reaction was allowed toproceed at 37° C. for 15 minutes. Thereafter, 50 μl of the reactionmixture was plated on ampicillin (final concentration: 50μg/ml)-containing LB agar medium, and cultured at 37° C. overnight. Asingle colony of transformed SOLR was recovered and cultured inampicillin (final concentration: 50 μg/ml)-containing LB medium at 37°C., followed by purification of plasmid DNA having an insert of interestusing QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).

The purified plasmid was subjected to an analysis of the entire sequenceof the insert by the primer walking method using the T3 primer describedin SEQ ID NO:5 and the T7 primer described in SEQ ID NO:6. By thissequence analysis, the gene sequence described in SEQ ID NO:38 wasobtained. Using the base sequence and the amino acid sequence of thisgene, homology search against known genes was carried out using ahomology search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As aresult, it was revealed that the obtained gene is the TRIP11 gene. Thehuman homologous factor of canine TRIP11 was human TRIP11 (homology:base sequence, 88%; amino acid sequence, 86%). The base sequence ofhuman TRIP11 is shown in SEQ ID NO:40, and the amino acid sequencethereof is shown in SEQ ID NO:41.

(4) Analysis of Expression in Each Tissue

The expression of the gene, which was obtained by the above-describedmethod, in normal tissues and various cell lines of dog and human wereinvestigated by the RT-PCR (Reverse Transcription-PCR) method. Thereverse transcription reaction was carried out as follows. That is,total RNA was extracted from 50 to 100 mg of each tissue or 5 to 10×10⁶cells of each cell line using TRIZOL reagent (manufactured byInvitrogen) in accordance with the protocol attached to the kit. Usingthis total RNA, cDNA was synthesized by Superscript First-StrandSynthesis System for RT-PCR (manufactured by Invitrogen) in accordancewith the protocol attached to the kit. As the cDNAs from human normaltissues (brain, hippocampus, testis, colon and placenta), Gene Pool cDNA(manufactured by Invitrogen), QUICK-Clone cDNA (manufactured byCLONTECH) and Large-Insert cDNA Library (manufactured by CLONTECH) wereused. The PCR reactions were carried out as follows using primers(described in SEQ ID NOs:42 and 43) specific to the obtained gene. Thatis, respective reagents and the attached buffer were mixed such that themixture should contain 0.25 μl of the sample prepared by the reversetranscription reaction, 2 μM each of the above primers, 0.2 mM each ofdNTP and 0.65 U of ExTaq polymerase (manufactured by Takara Shuzo Co.,Ltd.) in a total volume of 25 μl, and the reaction was carried out with30 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for1.5 minutes using Thermal Cycler (manufactured by BIO RAD). Theabove-described gene-specific primers were those which amplify theregions of the 1519th to 2957th bases of the base sequence of SEQ IDNO:38 (canine TRIP11 gene) and the 1872nd to 3310th bases of the basesequence of SEQ ID NO:40 (human TRIP11 gene), and can be used forinvestigation of the expression of both the canine TRIP11 gene and thehuman TRIP11 gene. As a control for comparison, primers (described inSEQ ID NOs:9 and 10) specific to GAPDH were used simultaneously. As aresult, as shown in FIG. 7, strong expression of the canine TRIP11 genewas observed in testis among the normal dog tissues, and on the otherhand, strong expression was observed in the canine breast cancer cellline. Expression of the human gene was confirmed, as is the case withthe canine TRIP11 gene, only in testis among the human normal tissues,but the expression was detected in many types of cancer cell lines suchas brain tumor, leukemia, breast cancer, lung cancer and esophaguscancer cell lines among human cancer cell lines. Thus, the human TRIP11gene was also confirmed to be specifically expressed in testis andcancer cells.

In FIG. 7, reference numeral 1 in the ordinate indicates the expressionpattern of the TRIP11 gene, and reference numeral 2 indicates theexpression pattern of the GAPDH gene as a control for comparison.

Example D-2 Preparation of Canine and Human TRIP11 Proteins (1)Preparation of Recombinant Protein

Based on the gene of SEQ ID NO:38 obtained in Example D-1, a recombinantprotein was prepared by the following method. NO: Respective reagentsand the attached buffer were mixed such that the mixture should contain1 μl of the vector which was prepared from the phagemid solutionobtained in Example D-1 and was subjected to the sequence analysis, 0.4μM each of two kinds of primers having SalI and XhoI restriction sites(described in SEQ ID NOs:44 and 45), 0.2 mM dNTP and 1.25 U of PrimeSTARHS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volumeof 50 μl, and PCR was carried out with 30 cycles of 98° C. for 10seconds, 55° C. for 5 seconds and 72° C. for 6 minutes using ThermalCycler (manufactured by BIO RAD). The above-described two kinds ofprimers were those which amplify the region encoding the entire aminoacid sequence of SEQ ID NO:39. After the PCR, the amplified DNA wassubjected to electrophoresis using 1% agarose gel, and a DNA fragment ofabout 6.0 kbp was purified using QIAquick Gel Extraction Kit(manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and plasmids were recovered thereafter, followed byconfirming, by sequencing, that the amplified gene fragment matches thesequence of interest. The plasmid that matched the sequence of interestwas treated with restriction enzymes SalI and XhoI and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30b (manufactured byNovagen) that had been treated with SalI and XhoI. Usage of this vectorenables production of a His-tag fusion recombinant protein. E. coli forexpression, BL21 (DE3), was transformed with this plasmid, andexpression of the protein of interest was induced in E. coli with 1 mMIPTG.

Further, based on the gene of SEQ ID NO:40, a recombinant protein of thehuman homologous gene was prepared by the following method. Respectivereagents and the attached buffer were mixed such that the mixture shouldcontain 1 μl of the cDNA prepared in Example D-1 whose expression couldbe confirmed by the RT-PCR method in various tissues/cells, 0.4 μM eachof two kinds of primers having NdeI and KpnI restriction sites(described in SEQ ID NOs:46 and 47), 0.2 mM dNTP and 1.25 U of PrimeSTARHS polymerase (manufactured by Takara Shuzo Co., Ltd.) in a total volumeof 50 μl, and PCR was carried out with 30 cycles of 98° C. for 10seconds, 55° C. for 5 seconds and 72° C. for 6 minutes using ThermalCycler (manufactured by BIO RAD). The above-described two kinds ofprimers were those which amplify the region encoding the entire aminoacid sequence of SEQ ID NO:41. After the PCR, the amplified DNA wassubjected to electrophoresis using 1% agarose gel, and a DNA fragment ofabout 6.0 kbp was purified using QIAquick Gel Extraction Kit(manufactured by QIAGEN).

The purified DNA fragment was ligated into a cloning vector pCR-Blunt(manufactured by Invitrogen). E. coli was transformed with the resultingligation product, and plasmids were recovered thereafter, followed byconfirming, by sequencing, that the amplified gene fragment matches thesequence of interest. The plasmid that matched the sequence of interestwas treated with restriction enzymes NdeI and KpnI and purified usingQIAquick Gel Extraction Kit, followed by inserting the gene sequence ofinterest into an expression vector for E. coli, pET30b (manufactured byNovagen) that had been treated with NdeI and KpnI. Usage of this vectorenables production of a His-tag fusion recombinant protein. E. coli forexpression, BL21 (DE3), was transformed with this plasmid, andexpression of the protein of interest was induced in E. coli with 1 mMIPTG.

(2) Purification of Recombinant Proteins

The above-obtained recombinant E. coli cells that expressed SEQ ID NO:38and SEQ ID NO:40, respectively, were cultured in 30 μg/mlkanamycin-containing LB medium at 37° C. until the absorbance at 600 nmreached about 0.7, and then isopropyl-β-D-1-thiogalactopyranoside wasadded thereto such that its final concentration should be 1 mM, followedby culturing them at 30° C. for 20 hours. Subsequently, the cells werecollected by centrifugation at 4,800 rpm for 10 minutes. The pellet ofthe cells was suspended in phosphate-buffered saline and furthersubjected to centrifugation at 4,800 rpm for 10 minutes to wash thecells.

The obtained pellet of E. coli cells was suspended in phosphate-bufferedsaline and subjected to sonication on ice. The sonicated solution of E.coli was centrifuged at 7,000 rpm for 15 minutes to obtain thesupernatant as the soluble fraction and the precipitate as the insolublefraction.

The insoluble fraction was suspended in 4% Triton X-100 solution and theresulting suspension was centrifuged at 7,000 rpm for 10 minutes. Thisoperation was repeated twice and an operation of removal of proteaseswas carried out. Thereafter, the residue was suspended inphosphate-buffered saline and an operation of removal of the surfactantwas carried out.

The residue was suspended in 6M guanidine hydrochloride-containing 20 mMphosphate buffer (pH 8.0), and the resulting suspension was left tostand at 4° C. for 20 hours to denature proteins. Thereafter, thesuspension was centrifuged at 7,000 rpm for 20 minutes, and the obtainedsoluble fraction was placed in a nickel chelate column prepared by aconventional method (carrier: Chelating Sepharose (trademark) Fast Flow(GE Health Care); column volume: 5 mL; equilibration buffer: 6Mguanidine hydrochloride-containing 20 mM phosphate buffer (pH 8.0)). Thefraction that was not adsorbed to the column was washed away with 10column volumes of 6 M sodium chloride-containing 20 mM phosphate buffer(pH 8.0) and 10 mM imidazole-containing 20 mM phosphate buffer (pH 8.0),and elution was immediately carried out with a four-step densitygradient of 50 mM-500 mM imidazole to obtain a purified fraction, whichwas used as the material for administration tests thereafter. Theproteins of interest in the eluted fractions were confirmed by Coomassiestaining carried out according to a conventional method. Among these,the canine TRIP11 protein is shown in FIG. 8.

To 1 ml of a reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl₂; pH7.4), 200 μl of the purified preparation obtained by the above-describedmethod was aliquoted, and 2 μl of enterokinase (manufactured by Novagen)was then added thereto, followed by leaving it to stand at roomtemperature overnight to cleave His tag. The resulting product waspurified using Enterokinase Cleavage Capture Kit (manufactured byNovagen) in accordance with the protocol attached to the kit.Subsequently, the buffer contained in 1.2 ml of the purified preparationobtained by the above-described method was replaced with physiologicalphosphate buffer (manufactured by Nissui Pharmaceutical) byultrafiltration using NANOSEP 10K OMEGA (manufactured by PALL), and theresulting solution was filtered aseptically using HT Tuffryn Acrodisc0.22 μm (manufactured by PALL) and used in the following experiments.

Example D-3 Test of Administration of Recombinant Protein toCancer-Bearing Dogs (1) Antitumor Assay

The anti-tumor effect of the two kinds of recombinant proteins whichwere purified as described above was assessed in two individuals ofcancer-bearing dogs having epidermal tumor (2 individuals having mammarygland tumor).

Therapeutic agents for a cancer(s) were prepared by mixing 0.5 ml ofFreund's incomplete adjuvant (manufactured by Wako Pure Chemicals) with100 μg (0.5 ml) of the recombinant canine TRIP11 and human TRIP11proteins, respectively, purified as described above. Each of theseagents was administered to a regional lymph node in the vicinity of thetumor a total of 3 times, by carrying out the subsequent administrations3 days and 7 days after the first administration. As a result, thetumors with a size of about 75 mm³ and 102 mm³, respectively, at thetime of administration of the therapeutic agents were reduced to 63 mm³and 85 mm³, respectively, 10 days after the first administration; 35 mm³and 42 mm³, respectively, 20 days after the first administration; and to15 mm³ and 19 mm³, respectively, 30 days after the first administrationof the therapeutic agent for a cancer(s).

Further, to a canine patient suffering from mastocytoma, a mixture of100 μg (0.5 ml) of the canine TRP11 protein with 0.5 ml of Freund'sincomplete adjuvant was administered a total of 3 times in the samemanner as described above. Concurrently with the respectiveadministrations, 100 μg of canine interleukin-12 was subcutaneouslyadministered. As a result, the tumor with a size of about 165 mm³ at thetime of administration of the therapeutic agent completely regressed 23days after the first administration of the therapeutic agent.

(2) Immune Inducibility Assay

Blood from the canine patient suffering from mammary gland tumor inwhich the anti-tumor effect was obtained in the administration test inthe above-described (1) was collected. Peripheral blood mononuclearcells were isolated according to a conventional method, and by theELISPOT assay for IFNγ using it, the immune inducibility of eachadministered protein was assayed.

In a 96-well plate manufactured by Millipore (MultiScreen-IP, MAIPS4510), 100 μL/well of 70% ethanol was placed and the plate was left tostand for 5 minutes, followed by removal of the ethanol by aspiration.The plate was washed with sterile water and 300 μl/well of 200 mM SodiumBicarbonate (pH8.2) was placed therein. After leaving it to stand for 5minutes, Sodium Bicarbonate was removed by aspiration, and then theplate was washed. Subsequently, 0.5 μg/well of anti-canine interferon γmonoclonal antibody (manufactured by R&D, clone 142529, MAB781) mixedwith 200 mM Sodium Bicarbonate was placed in wells, and the plate wasincubated at 37° C. overnight to immobilize the primary antibody. Afterremoval of the primary antibody by aspiration, 300 μL/well of a blockingsolution (1% BSA-5% sucrose-200 mM Sodium Bicarbonate (pH8.2)) was addedto the wells, and the plate was incubated at 4° C. overnight to blockthe plate. After removal of the blocking solution by aspiration, 300μL/well of 10% fetal calf serum-containing RPMI medium (manufactured byInvitrogen) was placed in the wells and the plate was left to stand for5 minutes, followed by removal of the medium by aspiration.Subsequently, 5×10⁵ cells/well of the canine peripheral bloodmononuclear cells suspended in 10% fetal calf serum-containing RPMImedium were placed in the plate, and 10 μL/well of the canine TRIP11 orthe human TRIP11 protein used in each administration was added thereto,followed by culturing the cells under the conditions of 37° C. and 5%CO₂ for 24 hours, to allow immunocytes that might exist in theperipheral blood mononuclear cells to produce interferon γ. After theculture, the medium was removed, and the wells were washed 6 times witha washing solution (0.1% Tween20-200 mM Sodium Bicarbonate (pH8.2)). Ineach well, 100 μL of rabbit anti-dog polyclonal antibody 1000-folddiluted with the above-described blocking solution was placed, and theplate was incubated at 4° C. overnight. After washing the wells 3 timeswith the above-described washing solution, 100 μL of HRP-labeledanti-rabbit antibody 1,000-fold diluted with the above-describedblocking solution was placed in each well, and the reaction was allowedto proceed at 37° C. for 2 hours. After washing the wells 3 times withthe above-described washing solution, the resultant was colored withKonica Immunostain (manufactured by Konica), and the wells were washedwith water to stop the reaction. Thereafter, the membrane was dried, andimage processing of the wells was carried out, followed by counting thenumber of spot-forming cells (SFC) using KS ELISPOT compact system (CarlZeiss, Inc., Germany).

As a result, in either canine patient to which the canine TRIP11 proteinor the human TRIP11 protein was administered, peripheral bloodmononuclear cells sampled before the administration showed no spots. Onthe other hand, in the canine patient to which the canine TRIP11 wasadministered, peripheral blood mononuclear cells sampled 10 days and 30days after the administration showed 26 and 65 spots, respectively. Inthe canine patient to which the human TRIP11 was administered,peripheral blood mononuclear cells sampled 10 days and 30 days after theadministration showed 31 and 72 spots, respectively.

From the above results, it is confirmed that immunocytes whichspecifically react with the administered recombinant protein and produceinterferon γ were induced in all of the canine patients to which therecombinant protein was administered, and it is thought that theanti-tumor effect described in the above-described (1) was exerted byimmunoreactions in which these immunocytes are mainly involved.

1. A method for inducing immunity, said method comprising administeringto an individual an effective amount of any one of the polypeptides (a)to (c) below, said polypeptide having an immunity-inducing activity: (a)a polypeptide consisting of not less than 7 consecutive amino acids ofthe amino acid sequence shown in SEQ ID NO: 26, 28 or 30 in SEQUENCELISTING; (b) a polypeptide having a homology of not less than 80% to thepolypeptide (a) and consisting of not less than 7 amino acids; and (c) apolypeptide comprising the polypeptide (a) or (b) as a partial sequencethereof.
 2. A method for inducing immunity, said method comprisingadministering to an individual an effective amount of a recombinantvector which comprises a polynucleotide encoding an immunity-inducingpolypeptide selected from any one of the polypeptides (a) to (c) below,said recombinant vector being capable of expressing said polypeptide invivo: (a) a polypeptide consisting of not less than 7 consecutive aminoacids of the amino acid sequence shown in SEQ ID NO: 26, 28 or 30 inSEQUENCE LISTING; (b) a polypeptide having a homology of not less than80% to the polypeptide (a) and consisting of not less than 7 aminoacids; and (c) a polypeptide comprising the polypeptide (a) or (b) as apartial sequence thereof.